U.S. patent application number 14/744847 was filed with the patent office on 2016-03-17 for viscosity-reducing excipient compounds for protein formulations.
The applicant listed for this patent is ReForm Biologics, LLC. Invention is credited to Rosa Casado Portilla, Robert P. Mahoney, Mark Moody, David S. Soane, Philip Wuthrich.
Application Number | 20160074515 14/744847 |
Document ID | / |
Family ID | 54936144 |
Filed Date | 2016-03-17 |
United States Patent
Application |
20160074515 |
Kind Code |
A1 |
Soane; David S. ; et
al. |
March 17, 2016 |
VISCOSITY-REDUCING EXCIPIENT COMPOUNDS FOR PROTEIN FORMULATIONS
Abstract
The invention encompasses formulations and methods for the
production thereof that permit the delivery of concentrated protein
solutions. The inventive methods can yield a lower viscosity liquid
formulation or a higher concentration of therapeutic or
nontherapeutic proteins in the liquid formulation, as compared to
traditional protein solutions.
Inventors: |
Soane; David S.; (Chestnut
Hill, MA) ; Wuthrich; Philip; (Belmont, MA) ;
Casado Portilla; Rosa; (Peabody, MA) ; Mahoney;
Robert P.; (Newbury, MA) ; Moody; Mark;
(Concord, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ReForm Biologics, LLC |
Cambridge |
MA |
US |
|
|
Family ID: |
54936144 |
Appl. No.: |
14/744847 |
Filed: |
June 19, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62014784 |
Jun 20, 2014 |
|
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|
62083623 |
Nov 24, 2014 |
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62136763 |
Mar 23, 2015 |
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Current U.S.
Class: |
424/130.1 ;
424/94.3; 435/188; 514/15.2; 530/363; 530/387.1 |
Current CPC
Class: |
A61K 9/0019 20130101;
A61K 47/18 20130101; A61K 47/12 20130101; A61K 47/42 20130101; C07K
16/00 20130101; C12N 9/2462 20130101; A61K 47/24 20130101; A61K
47/183 20130101; A61K 47/20 20130101; A61K 47/22 20130101; A61K
47/60 20170801; C12Y 302/01017 20130101; A61K 39/395 20130101; A61K
39/39591 20130101; C12N 9/96 20130101; A61K 38/47 20130101; A61K
38/385 20130101 |
International
Class: |
A61K 47/22 20060101
A61K047/22; A61K 47/18 20060101 A61K047/18; A61K 47/42 20060101
A61K047/42; C12N 9/36 20060101 C12N009/36; A61K 47/48 20060101
A61K047/48; A61K 38/47 20060101 A61K038/47; C12N 9/96 20060101
C12N009/96; A61K 39/395 20060101 A61K039/395; A61K 38/38 20060101
A61K038/38 |
Claims
1. A liquid formulation comprising a protein and an excipient
compound selected from the group consisting of hindered amines,
anionic aromatics, functionalized amino acids, oligopeptides,
short-chain organic acids, and low molecular weight aliphatic
polyacids, wherein the excipient compound is added in a
viscosity-reducing amount.
2. The liquid formulation of claim 1, wherein the protein is a
PEGylated protein and the excipient compound is a low molecular
weight aliphatic polyacid.
3. The liquid formulation of claim 1, wherein the formulation is a
pharmaceutical composition, and wherein the pharmaceutical
composition comprises a therapeutic protein, and wherein the
excipient compound is pharmaceutically acceptable excipient
compound.
4. The liquid formulation of claim 1, wherein the formulation is a
non-therapeutic formulation, and wherein the non-therapeutic
formulation comprises a non-therapeutic protein.
5. The formulation of claim 1, wherein the viscosity-reducing
amount reduces the viscosity of the formulation to a viscosity less
than the viscosity of a control formulation.
6. The formulation of claim 5, wherein the viscosity of the
formulation is at least about 10% less than the viscosity of the
control formulation.
7. (canceled)
8. The formulation of claim 6, wherein the viscosity of the
formulation is at least about 50% less than the viscosity of the
control formulation.
9. (canceled)
10. The formulation of claim 8, wherein the viscosity of the
formulation is at least about 90% less than the viscosity of the
control formulation.
11. The formulation of claim 5, wherein the viscosity is less than
about 100 cP.
12. The formulation of claim 5, wherein the viscosity is less than
about 50 cP.
13. The formulation of claim 5, wherein the viscosity is less than
about 20 cP.
14. The formulation of claim 5, wherein the viscosity is less than
about 10 cP.
15. The formulation of claim 1, wherein the excipient compound has
a molecular weight of <5000 Da.
16. (canceled)
17. The formulation of claim 15, wherein the excipient compound has
a molecular weight of <500 Da.
18. The formulation of claim 1, wherein the formulation contains at
least about 25 mg/mL of the protein.
19. (canceled)
20. The formulation of claim 18, wherein the formulation contains
at least about 200 mg/mL of the protein.
21. (canceled)
22. The formulation of claim 1, comprising between about 5 mg/mL to
about 300 mg/mL of the excipient compound.
23. (canceled)
24. The formulation of claim 22, comprising between about 20 mg/mL
to about 100 mg/mL.
25. (canceled)
26. The formulation of claim 1, wherein the formulation has an
improved stability when compared to the control formulation.
27. The formulation of claim 1, wherein the excipient compound is a
hindered amine.
28. The formulation of claim 27, wherein the hindered amine is
selected from the group consisting of caffeine, theophylline,
tyramine, imidazole, aspartame, saccharin, and acesulfame
potassium.
29. The formulation of claim 28, wherein the hindered amine is
caffeine.
30-33. (canceled)
34. The formulation of claim 31, wherein the hindered amine is
present in the formulation in an amount that is less than a
therapeutically effective amount.
35. (canceled)
36. The formulation of claim 35, wherein the second excipient
compound is selected from the group consisting of caffeine,
theophylline, tyramine, imidazole, aspartame, saccharin, and
acesulfame potassium.
37. (canceled)
38. A method of treating a disease or disorder in a mammal in need
thereof, comprising: administering to said mammal a liquid
therapeutic formulation, wherein the liquid therapeutic formulation
comprises a therapeutically effective amount of a therapeutic
protein and wherein the liquid therapeutic formulation further
comprises an pharmaceutically acceptable excipient compound
selected from the group consisting of hindered amines, anionic
aromatics, functionalized amino acids, oligopeptides, short-chain
organic acids, and low molecular weight aliphatic polyacids; and
wherein the therapeutic formulation is effective for the treatment
of the disorder.
39. The method of claim 38, wherein the therapeutic protein is a
PEGylated protein, and the excipient compound is a low molecular
weight aliphatic polyacid.
40. The method of claim 38, wherein the excipient compound is a
hindered amine.
41-48. (canceled)
49. A method of improving stability of a liquid protein
formulation, comprising: preparing a liquid protein formulation
comprising a therapeutic protein and an excipient compound selected
from the group selected from the group consisting of hindered
amines, anionic aromatics, functionalized amino acids,
oligopeptides, and short-chain organic acids, and low molecular
weight aliphatic polyacids, wherein the liquid protein formulation
demonstrates improved stability compared to a control liquid
protein formulation, wherein the control liquid protein formulation
does not contain the excipient compound and is otherwise identical
to the liquid protein formulation.
50. (canceled)
51. (canceled)
52. A liquid formulation comprising a protein and an excipient
compound selected from the group consisting of hindered amines,
anionic aromatics, functionalized amino acids, oligopeptides, and
short-chain organic acids, and low molecular weight aliphatic
polyacids, wherein the excipient compound results in improved
protein-protein interaction as measured by the protein diffusion
interaction parameter kD, or the second virial coefficient B22.
53. (canceled)
54. (canceled)
55. A method of improving a protein-related process comprising:
providing the liquid formulation of claim 1, and employing it in a
processing method.
56. (canceled)
57. (canceled)
58. The method of claim 55, wherein the processing method is
selected from the group consisting of filtration, pumping, mixing,
centrifugation, membrane separation, lyophilization, and
chromatography.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Ser. No. 62/014,784 filed Jun. 20, 2014, U.S.
Provisional Application No. 62/083,623, filed Nov. 24, 2014, and
U.S. Provisional Application Ser. No. 62/136,763 filed Mar. 23,
2015. The entire contents of the each of the above applications are
incorporated by reference herein.
FIELD OF APPLICATION
[0002] This application relates generally to formulations for
delivering biopolymers.
BACKGROUND
[0003] Biopolymers may be used for therapeutic or non-therapeutic
purposes. Biopolymer-based therapeutics, such as antibody or enzyme
formulations, are widely used in treating disease. Non-therapeutic
biopolymers, such as enzymes, peptides, and structural proteins,
have utility in non-therapeutic applications such as household,
nutrition, commercial, and industrial uses.
[0004] Biopolymers used in therapeutic applications must be
formulated to permit their introduction into the body for treatment
of disease. For example, it is advantageous to deliver antibody and
protein/peptide biopolymer formulations by subcutaneous (SC) or
intramuscular (IM) routes under certain circumstances, instead of
administering these formulations by intravenous (IV) injections. In
order to achieve better patient compliance and comfort with SC or
IM injection though, the liquid volume in the syringe is typically
limited to 2 to 3 ccs and the viscosity of the formulation is
typically lower than about 20 centipoise (cP) so that the
formulation can be delivered using conventional medical devices and
small-bore needles. These delivery parameters do not always fit
well with the dosage requirements for the formulations being
delivered.
[0005] Antibodies, for example, may need to be delivered at high
dose levels to exert their intended therapeutic effect. Using a
restricted liquid volume to deliver a high dose level of an
antibody formulation can require a high concentration of the
antibody in the delivery vehicle, sometimes exceeding a level of
150 mg/mL. At this dosage level, the viscosity-versus-concentration
plots of protein solutions lie beyond their linear-nonlinear
transition, such that the viscosity of the formulation rises
dramatically with increasing concentration. Increased viscosity,
however, is not compatible with standard SC or IM delivery systems.
The solutions of biopolymer-based therapeutics are also prone to
stability problems, such as precipitation, hazing, opalescence,
denaturing, and gel formation, reversible or irreversible
aggregation. The stability problems limit the shelf life of the
solutions or require special handling.
[0006] One approach to producing protein formulations for injection
is to transform the therapeutic protein solution into a powder that
can be reconstituted to form a suspension suitable for SC or IM
delivery. Lyophilization is a standard technique to produce protein
powders. Freeze-drying, spray drying and even precipitation
followed by super-critical-fluid extraction have been used to
generate protein powders for subsequent reconstitution. Powdered
suspensions are low in viscosity before re-dissolution (compared to
solutions at the same overall dose) and thus may be suitable for SC
or IM injection, provided the particles are sufficiently small to
fit through the needle. However, protein crystals that are present
in the powder have the inherent risk of triggering immune response.
The uncertain protein stability/activity following re-dissolution
poses further concerns. There remains a need in the art for
techniques to produce low viscosity protein formulations for
therapeutic purposes while avoiding the limitations introduced by
protein powder suspensions.
[0007] In addition to the therapeutic applications of proteins
described above, biopolymers such as enzymes, peptides, and
structural proteins can be used in non-therapeutic applications.
These non-therapeutic biopolymers can be produced from a number of
different pathways, for example, derived from plant sources, animal
sources, or produced from cell cultures.
[0008] The non-therapeutic proteins can be produced, transported,
stored, and handled as a granular or powdered material or as a
solution or dispersion, usually in water. The biopolymers for
non-therapeutic applications can be globular or fibrous proteins,
and certain forms of these materials can have limited solubility in
water or exhibit high viscosity upon dissolution. These solution
properties can present challenges to the formulation, handling,
storage, pumping, and performance of the non-therapeutic materials,
so there is a need for methods to reduce viscosity and improve
solubility and stability of non-therapeutic protein solutions.
[0009] Proteins are complex biopolymers, each with a uniquely
folded 3-D structure and surface energy map
(hydrophobic/hydrophilic regions and charges). In concentrated
protein solutions, these macromolecules may strongly interact and
even inter-lock in complicated ways, depending on their exact shape
and surface energy distribution. "Hot-spots" for strong specific
interactions lead to protein clustering, increasing solution
viscosity. To address these concerns, a number of excipient
compounds are used in biotherapeutic formulations, aiming to reduce
solution viscosity by impeding localized interactions and
clustering. These efforts are individually tailored, often
empirically, sometimes guided by in silico simulations.
Combinations of excipient compounds may be provided, but optimizing
such combinations again must progress empirically and on a case-by
case basis.
[0010] There remains a need in the art for a truly universal
approach to reducing viscosity in protein formulations at a given
concentration under nonlinear conditions. There is an additional
need in the art to achieve this viscosity reduction while
preserving the activity of the protein. It would be further
desirable to adapt the viscosity-reduction system to use with
formulations having tunable and sustained release profiles, and to
use with formulations adapted for depot injection.
SUMMARY OF THE INVENTION
[0011] Disclosed herein, in embodiments, are liquid formulations
comprising a protein and an excipient compound selected from the
group consisting of hindered amines, anionic aromatics,
functionalized amino acids, oligopeptides, short-chain organic
acids, and low molecular weight aliphatic polyacids, wherein the
excipient compound is added in a viscosity-reducing amount. In
embodiments, the protein is a PEGylated protein and the excipient
is a low molecular weight aliphatic polyacid. In embodiments, the
formulation is a pharmaceutical composition, and the therapeutic
formulation comprises a therapeutic protein, wherein the excipient
compound is a pharmaceutically acceptable excipient compound. In
embodiments, the formulation is a non-therapeutic formulation, and
the non-therapeutic formulation comprises a non-therapeutic
protein. In embodiments, the viscosity-reducing amount reduces
viscosity of the formulation to a viscosity less than the viscosity
of a control formulation. In embodiments, the viscosity of the
formulation is at least about 10% less than the viscosity of the
control formulation, or is at least about 30% less than the
viscosity of the control formulation, or is at least about 50% less
than the viscosity of the control formulation, or is at least about
70% less than the viscosity of the control formulation, or is at
least about 90% less than the viscosity of the control formulation.
In embodiments, the viscosity is less than about 100 cP, or is less
than about 50 cP, or is less than about 20 cP, or is less than
about 10 cP. In embodiments, the excipient compound has a molecular
weight of <5000 Da, or <1500 Da, or <500 Da. In
embodiments, the formulation contains at least about 25 mg/mL of
the protein, or at least about 100 mg/mL of the protein, or at
least about 200 mg/mL of the protein, or at least about 300 mg/mL
of the protein. In embodiments, the formulation comprises between
about 5 mg/mL to about 300 mg/mL of the excipient compound, or
comprises between about 10 mg/mL to about 200 mg/mL of the
excipient compound, or comprises between about 20 mg/mL to about
100 mg/mL, or comprises between about 25 mg/mL to about 75 mg/mL of
the excipient compound. In embodiments, the formulation has an
improved stability when compared to the control formulation. In
embodiments, the excipient compound is a hindered amine. In
embodiments, the hindered amine is selected from the group
consisting of caffeine, theophylline, tyramine, procaine,
lidocaine, imidazole, aspartame, saccharin, and acesulfame
potassium. In embodiments, the hindered amine is caffeine. In
embodiments, the hindered amine is a local injectable anesthetic
compound. The hindered amine can possess an independent
pharmacological property, and the hindered amine can be present in
the formulation in an amount that has an independent
pharmacological effect. In embodiments the hindered amine can be
present in the formulation in an amount that is less than a
therapeutically effective amount. The independent pharmacological
activity can be a local anesthetic activity. In embodiments, the
hindered amine possessing possessing the independent
pharmacological activity is combined with a second excipient
compound that further decreases the viscosity of the formulation.
The second excipient compound can be selected from the group
consisting of caffeine, theophylline, tyramine, procaine,
lidocaine, imidazole, aspartame, saccharin, and acesulfame
potassium. In embodiments, the formulation can comprise an
additional agent selected from the group consisting of
preservatives, surfactants, sugars, polysaccharides, arginine,
proline, hyaluronidase, stabilizers, and buffers.
[0012] Further disclosed herein are methods of treating a disease
or disorder in a mammal, comprising administering to said mammal a
liquid therapeutic formulation, wherein the therapeutic formulation
comprises a therapeutically effective amount of a therapeutic
protein, and wherein the formulation further comprises an
pharmaceutically acceptable excipient compound selected from the
group consisting of hindered amines, anionic aromatics,
functionalized amino acids, oligopeptides, short-chain organic
acids, and low molecular weight aliphatic polyacids; and wherein
the therapeutic formulation is effective for the treatment of the
disease or disorder. In embodiments, the therapeutic protein is a
PEGylated protein, and the excipient compound is a low molecular
weight aliphatic polyacid. In embodiments, the excipient is a
hindered amine. In embodiments, the hindered amine is a local
anesthetic compound. In embodiments, the formulation is
administered by subcutaneous injection, or an intramuscular
injection, or an intravenous injection. In embodiments, the
excipient compound is present in the therapeutic formulation in a
viscosity-reducing amount, and the viscosity-reducing amount
reduces viscosity of the therapeutic formulation to a viscosity
less than the viscosity of a control formulation. In embodiments,
the therapeutic formulation has an improved stability when compared
to the control formulation. In embodiments, the excipient compound
is essentially pure.
[0013] Further disclosed herein are methods of reducing pain at an
injection site of a therapeutic protein in a mammal in need
thereof, comprising: administering a liquid therapeutic formulation
by injection, wherein the therapeutic formulation comprises a
therapeutically effective amount of the therapeutic protein,
wherein the formulation further comprises an pharmaceutically
acceptable excipient compound selected from the group consisting of
local injectable anesthetic compounds, wherein the pharmaceutically
acceptable excipient compound is added to the formulation in a
viscosity-reducing amount; and wherein the mammal experiences less
pain with administration of the therapeutic formulation comprising
the excipient compound than that with administration of a control
therapeutic formulation, wherein the control therapeutic
formulation does not contain the excipient compound and is
otherwise identical to the therapeutic formulation.
[0014] Disclosed herein, in embodiments, are methods of improving
stability of a liquid protein formulation, comprising: preparing a
liquid protein formulation comprising a therapeutic protein and an
excipient compound selected from the group selected from the group
consisting of hindered amines, anionic aromatics, functionalized
amino acids, oligopeptides, and short-chain organic acids, and low
molecular weight aliphatic polyacids, wherein the liquid protein
formulation demonstrates improved stability compared to a control
liquid protein formulation, wherein the control liquid protein
formulation does not contain the excipient compound and is
otherwise identical to the liquid protein formulation. The
stability of the liquid formulation can be a cold storage
conditions stability, a room temperature stability or an elevated
temperature stability.
[0015] Also disclosed herein, in embodiments, are liquid
formulations comprising a protein and an excipient compound
selected from the group consisting of hindered amines, anionic
aromatics, functionalized amino acids, oligopeptides, short-chain
organic acids, and low molecular weight aliphatic polyacids,
wherein the presence of the excipient compound in the formulation
results in improved protein-protein interaction characteristics as
measured by the protein diffusion interaction parameter kD, or the
second virial coefficient B22. In embodiments, the formulation is a
therapeutic formulation, and comprises a therapeutic protein. In
embodiments, the formulation is a non-therapeutic formulation, and
comprises a non-therapeutic protein.
[0016] Further disclosed herein, in embodiments, are methods of
improving a protein-related process comprising providing the liquid
formulation described above, and employing it in a processing
method. In embodiments, the processing method includes filtration,
pumping, mixing, centrifugation, membrane separation,
lyophilization, or chromatography.
DETAILED DESCRIPTION
[0017] Disclosed herein are formulations and methods for their
production that permit the delivery of concentrated protein
solutions. In embodiments, the approaches disclosed herein can
yield a lower viscosity liquid formulation or a higher
concentration of therapeutic or nontherapeutic proteins in the
liquid formulation, as compared to traditional protein solutions.
In embodiments, the approaches disclosed herein can yield a liquid
formulation having improved stability when compared to a
traditional protein solution. A stable formulation is one in which
the protein contained therein substantially retains its physical
and chemical stability and its therapeutic or nontherapeutic
efficacy upon storage under storage conditions, whether cold
storage conditions, room temperature conditions, or elevated
temperature storage conditions. Advantageously, a stable
formulation can also offer protection against aggregation or
precipitation of the proteins dissolved therein. For example, the
cold storage conditions can entail storage in a refrigerator or
freezer. In some examples, cold storage conditions can entail
conventional refrigerator or freezer storage at a temperature of
10.degree. C. or less. In additional examples, the cold storage
conditions entail storage at a temperature from about 2.degree. to
about 10.degree. C. In other examples, the cold storage conditions
entail storage at a temperature of about 4.degree. C. In additional
examples, the cold storage conditions entail storage at freezing
temperature such as about 0.degree. C. or lower. In another
example, cold storage conditions entail storage at a temperature of
about -30.degree. C. to about 0.degree. C. The room temperature
storage conditions can entail storage at ambient temperatures, for
example, from about 10.degree. C. to about 30.degree. C. Elevated
temperature stability, for example, at temperatures from about
30.degree. C. to about 50.degree. C., can be used as part of an
accelerated aging study to predict the long term storage at typical
ambient (10-30.degree. C.) conditions.
[0018] It is well known to those skilled in the art of polymer
science and engineering that proteins in solution tend to form
entanglements, which can limit the translational mobility of the
entangled chains and interfere with the protein's therapeutic or
nontherapeutic efficacy. In embodiments, excipient compounds as
disclosed herein can suppress protein clustering due to specific
interactions between the excipient compound and the target protein
in solution. Excipient compounds as disclosed herein can be natural
or synthetic, and desirably are substances that the FDA generally
recognizes as safe ("GRAS").
1. DEFINITIONS
[0019] For the purpose of this disclosure, the term "protein"
refers to a sequence of amino acids having a chain length long
enough to produce a discrete tertiary structure, typically having a
molecular weight between 1-3000 kD. In some embodiments, the
molecular weight of the protein is between about 50-200 kD; in
other embodiments, the molecular weight of the protein is between
about 20-1000 kD or between about 20-2000 kD. In contrast to the
term "protein," the term "peptide" refers to a sequence of amino
acids that does not have a discrete tertiary structure. A wide
variety of biopolymers are included within the scope of the term
"protein." For example, the term "protein" can refer to therapeutic
or non-therapeutic proteins, including antibodies, aptamers, fusion
proteins, PEGylated proteins, synthetic polypeptides, protein
fragments, lipoproteins, enzymes, structural peptides, and the
like.
[0020] As non-limiting examples, therapeutic proteins can include
mammalian proteins such as hormones and prohormones (e.g., insulin
and proinsulin, glucagon, calcitonin, thyroid hormones (T3 or T4 or
thyroid-stimulating hormone), parathyroid hormone,
follicle-stimulating hormone, luteinizing hormone, growth hormone,
growth hormone releasing factor, and the like); clotting and
anti-clotting factors (e.g., tissue factor, von Willebrand's
factor, Factor VIIIC, Factor IX, protein C, plasminogen activators
(urokinase, tissue-type plasminogen activators), thrombin);
cytokines, chemokines, and inflammatory mediators; interferons;
colony-stimulating factors; interleukins (e.g., IL-1 through
IL-10); growth factors (e.g., vascular endothelial growth factors,
fibroblast growth factor, platelet-derived growth factor,
transforming growth factor, neurotrophic growth factors,
insulin-like growth factor, and the like); albumins; collagens and
elastins; hematopoietic factors (e.g., erythropoietin,
thrombopoietin, and the like); osteoinductive factors (e.g., bone
morphogenetic protein); receptors (e.g., integrins, cadherins, and
the like); surface membrane proteins; transport proteins;
regulatory proteins; antigenic proteins (e.g., a viral component
that acts as an antigen); and antibodies. The term "antibody" is
used herein in its broadest sense, to include as non-limiting
examples monoclonal antibodies (including, for example, full-length
antibodies with an immunoglobulin Fc region), single-chain
molecules, bi-specific and multi-specific antibodies, diabodies,
antibody compositions having polyepitopic specificity, and
fragments of antibodies (including, for example, Fab, Fv, and
F(ab')2). Antibodies can also be termed "immunoglobulins." An
antibody is understood to be directed against a specific protein or
non-protein "antigen," which is a biologically important material;
the administration of a therapeutically effective amount of an
antibody to a patient can complex with the antigen, thereby
altering its biological properties so that the patient experiences
a therapeutic effect.
[0021] In embodiments, the proteins are PEGylated, meaning that
they comprise poly(ethylene glycol) ("PEG") and/or polypropylene
glycol) ("PPG") units. PEGylated proteins, or PEG-protein
conjugates, have found utility in therapeutic applications due to
their beneficial properties such as solubility, pharmacokinetics,
pharmacodynamics, immunogenicity, renal clearance, and stability.
Non-limiting examples of PEGylated proteins are PEGylated
interferons (PEG-IFN), PEGylated anti-VEGF, PEG protein conjugate
drugs, Adagen, Pegaspargase, Pegfilgrastim, Pegloticase,
Pegvisomant, PEGylated epoetin-.beta., and Certolizumab pegol.
[0022] PEGylated proteins can be synthesized by a variety of
methods such as a reaction of protein with a PEG reagent having one
or more reactive functional groups. The reactive functional groups
on the PEG reagent can form a linkage with the protein at targeted
protein sites such as lysine, histidine, cysteine, and the
N-terminus. Typical PEGylation reagents have reactive functional
groups such as aldehyde, maleimide, or succinimide groups that have
specific reactivity with targeted amino acid residues on proteins.
The PEGylation reagents can have a PEG chain length from about 1 to
about 1000 PEG and/or PPG repeating units. Other methods of
PEGylation include glyco-PEGylation, where the protein is first
glycosylated and then the glycosylated residues are PEGylated in a
second step. Certain PEGylation processes are assisted by enzymes
like sialyltransferase and transglutaminase.
[0023] While the PEGylated proteins can offer therapeutic
advantages over native, non-PEGylated proteins, these materials can
have physical or chemical properties that make them difficult to
purify, dissolve, filter, concentrate, and administer. The
PEGylation of a protein can lead to a higher solution viscosity
compared to the native protein, and this generally requires the
formulation of PEGylated protein solutions at lower
concentrations.
[0024] It is desirable to formulate protein therapeutics in stable,
low viscosity solutions so they can be administered to patients in
a minimal injection volume. For example, the subcutaneous (SC) or
intramuscular (IM) injection of drugs generally requires a small
injection volume, preferably less than 2 mL. The SC and IM
injection routes are well suited to self-administered care, and
this is a less costly and more accessible form of treatment
compared with intravenous (IV) injection which is only conducted
under direct medical supervision. Formulations for SC or IM
injection require a low solution viscosity, generally below 30 cP,
and preferably below 20 cP, to allow easy flow of the therapeutic
solution through a narrow gauge needle. This combination of small
injection volume and low viscosity requirements present a challenge
to the use of PEGylated protein therapeutics in SC or IM injection
routes.
[0025] Those proteins having therapeutic effects may be termed
"therapeutic proteins"; formulations containing therapeutic
proteins in therapeutically effective amounts may be termed
"therapeutic formulations." The therapeutic protein contained in a
therapeutic formulation may also be termed its "protein active
ingredient." Typically, a therapeutic formulation comprises a
therapeutically effective amount of a protein active ingredient and
an excipient, with or without other optional components. As used
herein, the term "therapeutic" includes both treatments of existing
disorders and preventions of disorders.
[0026] A "treatment" includes any measure intended to cure, heal,
alleviate, improve, remedy, or otherwise beneficially affect the
disorder, including preventing or delaying the onset of symptoms
and/or alleviating or ameliorating symptoms of the disorder. Those
patients in need of a treatment include both those who already have
a specific disorder, and those for whom the prevention of a
disorder is desirable. A disorder is any condition that alters the
homeostatic wellbeing of a mammal, including acute or chronic
diseases, or pathological conditions that predispose the mammal to
an acute or chronic disease. Non-limiting examples of disorders
include cancers, metabolic disorders (e.g., diabetes), allergic
disorders (e.g., asthma), dermatological disorders, cardiovascular
disorders, respiratory disorders, hematological disorders,
musculoskeletal disorders, inflammatory or rheumatological
disorders, autoimmune disorders, gastrointestinal disorders,
urological disorders, sexual and reproductive disorders,
neurological disorders, and the like. The term "mammal" for the
purposes of treatment can refer to any animal classified as a
mammal, including humans, domestic animals, pet animals, farm
animals, sporting animals, working animals, and the like. A
"treatment" can therefore include both veterinary and human
treatments. For convenience, the mammal undergoing such "treatment"
can be referred to as a "patient." In certain embodiments, the
patient can be of any age, including fetal animals in utero.
[0027] In embodiments, a treatment involves providing a
therapeutically effective amount of a therapeutic formulation to a
mammal in need thereof. A "therapeutically effective amount" is at
least the minimum concentration of the therapeutic protein
administered to the mammal in need thereof, to effect a treatment
of an existing disorder or a prevention of an anticipated disorder
(either such treatment or such prevention being a "therapeutic
intervention"). Therapeutically effective amounts of various
therapeutic proteins that may be included as active ingredients in
the therapeutic formulation may be familiar in the art; or, for
therapeutic proteins discovered or applied to therapeutic
interventions hereinafter, the therapeutically effective amount can
be determined by standard techniques carried out by those having
ordinary skill in the art, using no more than routine
experimentation.
[0028] Those proteins used for non-therapeutic purposes (i.e.,
purposes not involving treatments), such as household, nutrition,
commercial, and industrial applications, may be termed
"non-therapeutic proteins." Formulations containing non-therapeutic
proteins may be termed "non-therapeutic formulations". The
non-therapeutic proteins can be derived from plant sources, animal
sources, or produced from cell cultures; they also can be enzymes
or structural proteins. The non-therapeutic proteins can be used in
in household, nutrition, commercial, and industrial applications
such as catalysts, human and animal nutrition, processing aids,
cleaners, and waste treatment.
[0029] An important category of non-therapeutic biopolymers is
enzymes. Enzymes have a number of non-therapeutic applications, for
example, as catalysts, human and animal nutritional ingredients,
processing aids, cleaners, and waste treatment agents. Enzyme
catalysts are used to accelerate a variety of chemical reactions.
Examples of enzyme catalysts for non-therapeutic uses include
catalases, oxidoreductases, transferases, hydrolases, lyases,
isomerases, and ligases. Human and animal nutritional uses of
enzymes include nutraceuticals, nutritive sources of protein,
chelation or controlled delivery of micronutrients, digestion aids,
and supplements; these can be derived from amylase, protease,
trypsin, lactase, and the like. Enzymatic processing aids are used
to improve the production of food and beverage products in
operations like baking, brewing, fermenting, juice processing, and
winemaking Examples of these food and beverage processing aids
include amylases, cellulases, pectinases, glucanases, lipases, and
lactases. Enzymes can also be used in the production of biofuels.
Ethanol for biofuels, for example, can be aided by the enzymatic
degradation of biomass feedstocks such as cellulosic and
lignocellulosic materials. The treatment of such feedstock
materials with cellulases and ligninases transforms the biomass
into a substrate that can be fermented into fuels. In other
commercial applications, enzymes are used as detergents, cleaners,
and stain lifting aids for laundry, dish washing, surface cleaning,
and equipment cleaning applications. Typical enzymes for this
purpose include proteases, cellulases, amylases, and lipases. In
addition, non-therapeutic enzymes are used in a variety of
commercial and industrial processes such as textile softening with
cellulases, leather processing, waste treatment, contaminated
sediment treatment, water treatment, pulp bleaching, and pulp
softening and debonding. Typical enzymes for these purposes are
amylases, xylanases, cellulases, and ligninases.
[0030] Other examples of non-therapeutic biopolymers include
fibrous or structural proteins such as keratins, collagen, gelatin,
elastin, fibroin, actin, tubulin, or the hydrolyzed, degraded, or
derivatized forms thereof. These materials are used in the
preparation and formulation of food ingredients such as gelatin,
ice cream, yogurt, and confections; they area also added to foods
as thickeners, rheology modifiers, mouthfeel improvers, and as a
source of nutritional protein. In the cosmetics and personal care
industry, collagen, elastin, keratin, and hydrolyzed keratin are
widely used as ingredients in skin care and hair care formulations.
Still other examples of non-therapeutic biopolymers are whey
proteins such as beta-lactoglobulin, alpha-lactalbumin, and serum
albumin. These whey proteins are produced in mass scale as a
byproduct from dairy operations and have been used for a variety of
non-therapeutic applications.
2. THERAPEUTIC FORMULATIONS
[0031] In one aspect, the formulations and methods disclosed herein
provide stable liquid formulations of improved or reduced
viscosity, comprising a therapeutic protein in a therapeutically
effective amount and an excipient compound. In embodiments, the
formulation can improve the stability while providing an acceptable
concentration of active ingredients and an acceptable viscosity. In
embodiments, the formulation provides an improvement in stability
when compared to a control formulation; for the purposes of this
disclosure, a control formulation is a formulation containing the
protein active ingredient that is identical on a dry weight basis
in every way to the therapeutic formulation except that it lacks
the excipient compound. In embodiments, improved stability of the
protein containing formulation is in the form of lower percentage
of soluble aggregates, particulates, subvisible particles, or gel
formation, compared to a control formulation.
[0032] It is understood that the viscosity of a liquid protein
formulation can be affected by a variety of factors, including but
not limited to: the nature of the protein itself (e.g., enzyme,
antibody, receptor, fusion protein, etc.); its size,
three-dimensional structure, chemical composition, and molecular
weight; its concentration in the formulation; the components of the
formulation besides the protein; the desired pH range; the storage
conditions for the formulation; and the method of administering the
formulation to the patient. Therapeutic proteins most suitable for
use with the excipient compounds described herein are preferably
essentially pure, i.e., free from contaminating proteins. In
embodiments, an "essentially pure" therapeutic protein is a protein
composition comprising at least 90% by weight of the therapeutic
protein, or preferably at least 95% by weight, or more preferably,
at least 99% by weight, all based on the total weight of
therapeutic proteins and contaminating proteins in the composition.
For the purposes of clarity, a protein added as an excipient is not
intended to be included in this definition. The therapeutic
formulations described herein are intended for use as
pharmaceutical-grade formulations, i.e., formulations intended for
use in treating a mammal, in such a form that the desired
therapeutic efficacy of the protein active ingredient can be
achieved, and without containing components that are toxic to the
mammal to whom the formulation is to be administered.
[0033] In embodiments, the therapeutic formulation contains at
least 25 mg/mL of protein active ingredient. In other embodiments,
the therapeutic formulation contains at least 100 mg/mL of protein
active ingredient. In other embodiments, the therapeutic
formulation contains at least 200 mg/mL of protein active
ingredient. In yet other embodiments, the therapeutic formulation
solution contains at least 300 mg/mL of protein active ingredient.
Generally, the excipient compounds disclosed herein are added to
the therapeutic formulation in an amount between about 5 to about
300 mg/mL. In embodiments, the excipient compound can be added in
an amount of about 10 to about 200 mg/mL. In embodiments, the
excipient compound can be added in an amount of about 20 to about
100 mg/mL. In embodiments, the excipient can be added in an amount
of about 25 to about 75 mg/mL.
[0034] Excipient compounds of various molecular weights are
selected for specific advantageous properties when combined with
the protein active ingredient in a formulation. Examples of
therapeutic formulations comprising excipient compounds are
provided below. In embodiments, the excipient compound has a
molecular weight of <5000 Da. In embodiments, the excipient
compound has a molecular weight of <1000 Da. In embodiments, the
excipient compound has a molecular weight of <500 Da.
[0035] In embodiments, the excipient compounds disclosed herein is
added to the therapeutic formulation in a viscosity-reducing
amount. In embodiments, a viscosity-reducing amount is the amount
of an excipient compound that reduces the viscosity of the
formulation at least 10% when compared to a control formulation;
for the purposes of this disclosure, a control formulation is a
formulation containing the protein active ingredient that is
identical on a dry weight basis in every way to the therapeutic
formulation except that it lacks the excipient compound. In
embodiments, the viscosity-reducing amount is the amount of an
excipient compound that reduces the viscosity of the formulation at
least 30% when compared to the control formulation. In embodiments,
the viscosity-reducing amount is the amount of an excipient
compound that reduces the viscosity of the formulation at least 50%
when compared to the control formulation. In embodiments, the
viscosity-reducing amount is the amount of an excipient compound
that reduces the viscosity of the formulation at least 70% when
compared to the control formulation. In embodiments, the
viscosity-reducing amount is the amount of an excipient compound
that reduces the viscosity of the formulation at least 90% when
compared to the control formulation.
[0036] In embodiments, the viscosity-reducing amount yields a
therapeutic formulation having a viscosity of less than 100 cP. In
other embodiments, the therapeutic formulation has a viscosity of
less than 50 cP. In other embodiments, the therapeutic formulation
has a viscosity of less than 20 cP. In yet other embodiments, the
therapeutic formulation has a viscosity of less than 10 cP. The
term "viscosity" as used herein refers to a dynamic viscosity value
when measured by the methods disclosed herein.
[0037] Therapeutic formulations in accordance with this disclosure
have certain advantageous properties. In embodiments, the
therapeutic formulations are resistant to shear degradation, phase
separation, clouding out, precipitation, and denaturing. In
embodiments, the therapeutic formulations are processed, purified,
stored, syringed, dosed, filtered, and centrifuged more
effectively, compared with a control formulation. In embodiments,
the therapeutic formulations are administered to a patient at high
concentration of therapeutic protein. In embodiments, the
therapeutic formulations are administered to patients with less
discomfort than would be experienced with a similar formulation
lacking the therapeutic excipient. In embodiments, the therapeutic
formulations are administered as a depot injection. In embodiments,
the therapeutic formulations extend the half-life of the
therapeutic protein in the body. These features of therapeutic
formulations as disclosed herein would permit the administration of
such formulations by intramuscular or subcutaneous injection in a
clinical situation, i.e., a situation where patient acceptance of
an intramuscular injection would include the use of small-bore
needles typical for IM/SC purposes and the use of a tolerable (for
example, 2-3 cc) injected volume, and where these conditions result
in the administration of an effective amount of the formulation in
a single injection at a single injection site. By contrast,
injection of a comparable dosage amount of the therapeutic protein
using conventional formulation techniques would be limited by the
higher viscosity of the conventional formulation, so that a SC/IM
injection of the conventional formulation would not be suitable for
a clinical situation.
[0038] In embodiments, the therapeutic excipient has antioxidant
properties that stabilize the therapeutic protein against oxidative
damage. In embodiments, the therapeutic formulation is stored at
ambient temperatures, or for extended time at refrigerator
conditions without appreciable loss of potency for the therapeutic
protein. In embodiments, the therapeutic formulation is dried down
for storage until it is needed; then it is reconstituted with an
appropriate solvent, e.g., water. Advantageously, the formulations
prepared as described herein can be stable over a prolonged period
of time, from months to years. When exceptionally long periods of
storage are desired, the formulations can be preserved in a freezer
(and later reactivated) without fear of protein denaturation. In
embodiments, formulations can be prepared for long-term storage
that do not require refrigeration.
[0039] Methods for preparing therapeutic formulations may be
familiar to skilled artisans. The therapeutic formulations of the
present invention can be prepared, for example, by adding the
excipient compound to the formulation before or after the
therapeutic protein is added to the solution. The therapeutic
formulation can, for example, be produced by combining the
therapeutic protein and the excipient at a first (lower)
concentration and then processed by filtration or centrifugation to
produce a second (higher) concentration of the therapeutic protein.
Therapeutic formulations can be made with one or more of the
excipient compounds with chaotropes, kosmotropes, hydrotropes, and
salts. Therapeutic formulations can be made with one or more of the
excipient compounds using techniques such as encapsulation,
dispersion, liposome, vesicle formation, and the like. Methods for
preparing therapeutic formulations comprising the excipient
compounds disclosed herein can include combinations of the
excipient compounds. In embodiments, combinations of excipients can
produce benefits in lower viscosity, improved stability, or reduced
injection site pain. Other additives may be introduced into the
therapeutic formulations during their manufacture, including
preservatives, surfactants, sugars, sucrose, trehalose,
polysaccharides, arginine, proline, hyaluronidase, stabilizers,
buffers, and the like. As used herein, a pharmaceutically
acceptable excipient compound is one that is non-toxic and suitable
for animal and/or human administration.
3. NON-THERAPEUTIC FORMULATIONS
[0040] In one aspect, the formulations and methods disclosed herein
provide stable liquid formulations of improved or reduced
viscosity, comprising a non-therapeutic protein in an effective
amount and an excipient compound. In embodiments, the formulation
improves the stability while providing an acceptable concentration
of active ingredients and an acceptable viscosity. In embodiments,
the formulation provides an improvement in stability when compared
to a control formulation; for the purposes of this disclosure, a
control formulation is a formulation containing the protein active
ingredient that is identical on a dry weight basis in every way to
the non-therapeutic formulation except that it lacks the excipient
compound.
[0041] It is understood that the viscosity of a liquid protein
formulation can be affected by a variety of factors, including but
not limited to: the nature of the protein itself (e.g., enzyme,
structural protein, degree of hydrolysis, etc.); its size,
three-dimensional structure, chemical composition, and molecular
weight; its concentration in the formulation; the components of the
formulation besides the protein; the desired pH range; and the
storage conditions for the formulation.
[0042] In embodiments, the non-therapeutic formulation contains at
least 25 mg/mL of protein active ingredient. In other embodiments,
the non-therapeutic formulation contains at least 100 mg/mL of
protein active ingredient. In other embodiments, the
non-therapeutic formulation contains at least 200 mg/mL of protein
active ingredient. In yet other embodiments, the non-therapeutic
formulation solution contains at least 300 mg/mL of protein active
ingredient. Generally, the excipient compounds disclosed herein are
added to the non-therapeutic formulation in an amount between about
5 to about 300 mg/mL. In embodiments, the excipient compound is
added in an amount of about 10 to about 200 mg/mL. In embodiments,
the excipient compound is added in an amount of about 20 to about
100 mg/mL. In embodiments, the excipient is added in an amount of
about 25 to about 75 mg/mL.
[0043] Excipient compounds of various molecular weights are
selected for specific advantageous properties when combined with
the protein active ingredient in a formulation. Examples of
non-therapeutic formulations comprising excipient compounds are
provided below. In embodiments, the excipient compound has a
molecular weight of <5000 Da. In embodiments, the excipient
compound has a molecular weight of <1000 Da. In embodiments, the
excipient compound has a molecular weight of <500 Da.
[0044] In embodiments, the excipient compounds disclosed herein is
added to the non-therapeutic formulation in a viscosity-reducing
amount. In embodiments, a viscosity-reducing amount is the amount
of an excipient compound that reduces the viscosity of the
formulation at least 10% when compared to a control formulation;
for the purposes of this disclosure, a control formulation is a
formulation containing the protein active ingredient that is
identical on a dry weight basis in every way to the therapeutic
formulation except that it lacks the excipient compound. In
embodiments, the viscosity-reducing amount is the amount of an
excipient compound that reduces the viscosity of the formulation at
least 30% when compared to the control formulation. In embodiments,
the viscosity-reducing amount is the amount of an excipient
compound that reduces the viscosity of the formulation at least 50%
when compared to the control formulation. In embodiments, the
viscosity-reducing amount is the amount of an excipient compound
that reduces the viscosity of the formulation at least 70% when
compared to the control formulation. In embodiments, the
viscosity-reducing amount is the amount of an excipient compound
that reduces the viscosity of the formulation at least 90% when
compared to the control formulation.
[0045] In embodiments, the viscosity-reducing amount yields a
non-therapeutic formulation having a viscosity of less than 100 cP.
In other embodiments, the non-therapeutic formulation has a
viscosity of less than 50 cP. In other embodiments, the
non-therapeutic formulation has a viscosity of less than 20 cP. In
yet other embodiments, the non-therapeutic formulation has a
viscosity of less than 10 cP. The term "viscosity" as used herein
refers to a dynamic viscosity value.
[0046] Non-therapeutic formulations in accordance with this
disclosure can have certain advantageous properties. In
embodiments, the non-therapeutic formulations are resistant to
shear degradation, phase separation, clouding out, precipitation,
and denaturing. In embodiments, the therapeutic formulations can be
processed, purified, stored, pumped, filtered, and centrifuged more
effectively, compared with a control formulation.
[0047] In embodiments, the non-therapeutic excipient has
antioxidant properties that stabilize the non-therapeutic protein
against oxidative damage. In embodiments, the non-therapeutic
formulation is stored at ambient temperatures, or for extended time
at refrigerator conditions without appreciable loss of potency for
the non-therapeutic protein. In embodiments, the non-therapeutic
formulation is dried down for storage until it is needed; then it
can be reconstituted with an appropriate solvent, e.g., water.
Advantageously, the formulations prepared as described herein is
stable over a prolonged period of time, from months to years. When
exceptionally long periods of storage are desired, the formulations
are preserved in a freezer (and later reactivated) without fear of
protein denaturation. In embodiments, formulations are prepared for
long-term storage that do not require refrigeration.
[0048] Methods for preparing non-therapeutic formulations
comprising the excipient compounds disclosed herein may be familiar
to skilled artisans. For example, the excipient compound can be
added to the formulation before or after the non-therapeutic
protein is added to the solution. The non-therapeutic formulation
can be produced at a first (lower) concentration and then processed
by filtration or centrifugation to produce a second (higher)
concentration. Non-therapeutic formulations can be made with one or
more of the excipient compounds with chaotropes, kosmotropes,
hydrotropes, and salts. Non-therapeutic formulations can be made
with one or more of the excipient compounds using techniques such
as encapsulation, dispersion, liposome, vesicle formation, and the
like. Other additives can be introduced into the non-therapeutic
formulations during their manufacture, including preservatives,
surfactants, stabilizers, and the like.
4. EXCIPIENT COMPOUNDS
[0049] Several excipient compounds are described herein, each
suitable for use with one or more therapeutic or non-therapeutic
proteins, and each allowing the formulation to be composed so that
it contains the protein(s) at a high concentration. Some of the
categories of excipient compounds described below are: (1) hindered
amines; (2) anionic aromatics; (3) functionalized amino acids; and
(4) oligopeptides. Without being bound by theory, the excipient
compounds described herein are thought to associate with certain
fragments, sequences, structures, or sections of a therapeutic
protein that otherwise would be involved in inter-particle (i.e.,
protein-protein) interactions. The association of these excipient
compounds with the therapeutic or non-therapeutic protein can mask
the inter-protein interactions such that the proteins can be
formulated in high concentration without causing excessive solution
viscosity. Excipient compounds advantageously can be water-soluble,
therefore suitable for use with aqueous vehicles. In embodiments,
the excipient compounds have a water solubility of >10 mg/mL. In
embodiments, the excipient compounds have a water solubility of
>100 mg/mL. In embodiments, the excipient compounds have a water
solubility of >500 mg/mL. Advantageously for therapeutic
proteins, the excipient compounds can be derived from materials
that are biologically acceptable and are non-immunogenic, and are
thus suitable for pharmaceutical use. In therapeutic embodiments,
the excipient compounds can be metabolized in the body to yield
biologically compatible and non-immunogenic byproducts.
[0050] a. Excipient Compound Category 1: Hindered Amines
[0051] High concentration solutions of therapeutic or
non-therapeutic proteins can be formulated with hindered amine
small molecules as excipient compounds. As used herein, the term
"hindered amine" refers to a small molecule containing at least one
bulky or sterically hindered group, consistent with the examples
below. Hindered amines can be used in the free base form, in the
protonated form, or a combination of the two. In protonated forms,
the hindered amines can be associated with an anionic counterion
such as chloride, hydroxide, bromide, iodide, fluoride, acetate,
formate, phosphate, sulfate, or carboxylate. Hindered amine
compounds useful as excipient compounds can contain secondary
amine, tertiary amine, quaternary ammonium, pyridinium,
pyrrolidone, pyrrolidine, piperidine, morpholine, or guanidinium
groups, such that the excipient compound has a cationic charge in
aqueous solution at neutral pH. The hindered amine compounds also
contain at least one bulky or sterically hindered group, such as
cyclic aromatic, cycloaliphatic, cyclohexyl, or alkyl groups. In
embodiments, the sterically hindered group can itself be an amine
group such as a dialkylamine, trialkylamine, guanidinium,
pyridinium, or quaternary ammonium group. Without being bound by
theory, the hindered amine compounds are thought to associate with
aromatic sections of the proteins such as phenylalanine,
tryptophan, and tyrosine, by a cation pi interaction. In
embodiments, the cationic group of the hindered amine can have an
affinity for the electron rich pi structure of the aromatic amino
acid residues in the protein, so that they can shield these
sections of the protein, thereby decreasing the tendency of such
shielded proteins to associate and agglomerate.
[0052] In embodiments, the hindered amine excipient compounds has a
chemical structure comprising imidazole, imidazoline, or
imidazolidine groups, or salts thereof, such as imidazole,
1-methylimidazole, 4-methylimidazole, 1-hexyl-3-methylimidazolium
chloride, histamine, 4-methylhistamine, alpha-methylhistamine,
betahistine, beta-alanine, 2-methyl-2-imidazoline,
1-butyl-3-methylimidazolium chloride, uric acid, potassium urate,
betazole, carnosine, aspartame, saccharin, acesulfame potassium,
xanthine, theophylline, theobromine, caffeine, and anserine. In
embodiments, the hindered amine excipient compounds is selected
from the group consisting of dimethylethanolamine,
dimethylaminopropylamine, triethanolamine, dimethylbenzylamine,
dimethylcyclohexylamine, diethylcyclohexylamine,
dicyclohexylmethylamine, hexamethylene biguanide,
poly(hexamethylene biguanide), imidazole, dimethylglycine,
agmatine, diazabicyclo[2.2.2]octane, tetramethylethylenediamine,
N,N-dimethylethanolamine, ethanolamine phosphate, glucosamine,
choline chloride, phosphocholine, niacinamide, isonicotinamide,
N,N-diethyl nicotinamide, nicotinic acid sodium salt, tyramine,
3-aminopyridine, 2,4,6-trimethylpyridine, 3-pyridine methanol,
nicotinamide adenosine dinucleotide, biotin, morpholine,
N-methylpyrrolidone, 2-pyrrolidinone, procaine, lidocaine,
dicyandiamide-taurine adduct, 2-pyridylethylamine,
dicyandiamide-benzyl amine adduct, dicyandiamide-alkylamine adduct,
dicyandiamide-cycloalkylamine adduct, and
dicyandiamide-aminomethanephosphonic acid adducts. In embodiments,
a hindered amine compound consistent with this disclosure is
formulated as a protonated ammonium salt. In embodiments, a
hindered amine compound consistent with this disclosure is
formulated as a salt with an inorganic anion or organic anion as
the counterion. In embodiments, high concentration solutions of
therapeutic or non-therapeutic proteins are formulated with a
combination of caffeine with a benzoic acid, a hydroxybenzoic acid,
or a benzenesulfonic acid as excipient compounds. In embodiments,
the hindered amine excipient compounds is metabolized in the body
to yield biologically compatible byproducts. In some embodiments,
the hindered amine excipient compound is present in the formulation
at a concentration of about 250 mg/ml or less. In additional
embodiments, the hindered amine excipient compound is present in
the formulation at a concentration of about 10 mg/ml to about 200
mg/ml. In yet additional aspects, the hindered amine excipient
compound is present in the formulation at a concentration of about
20 to about 120 mg/ml.
[0053] In embodiments, certain hindered amine excipient compounds
can possess other pharmacological properties. As examples,
xanthines are a category of hindered amines having independent
pharmacological properties, including stimulant properties and
bronchodilator properties when systemically absorbed.
Representative xanthines include caffeine, aminophylline,
3-isobutyl-1-methylxanthine, paraxanthine, pentoxifylline,
theobromine, theophylline, and the like. Methylated xanthines are
understood to affect force of cardiac contraction, heart rate, and
bronchodilation. In some embodiments, the xanthine excipient
compound is present in the formulation at a concentration of about
30 mg/ml or less.
[0054] Another category of hindered amines having independent
pharmacological properties are the local injectable anesthetic
compounds. Local injectable anesthetic compounds are hindered
amines that have a three-component molecular structure of (a) a
lipophilic aromatic ring, (b) an intermediate ester or amide
linkage, and (c) a secondary or tertiary amine. This category of
hindered amines is understood to interrupt neural conduction by
inhibiting the influx of sodium ions, thereby inducing local
anesthesia. The lipophilic aromatic ring for a local anesthetic
compound may be formed of carbon atoms (e.g., a benzene ring) or it
may comprise heteroatoms (e.g., a thiophene ring). Representative
local injectable anesthetic compounds include, but are not limited
to, amylocaine, articaine, bupivicaine, butacaine, butanilicaine,
chlorprocaine, cocaine, cyclomethycaine, dimethocaine, editocaine,
hexylcaine, isobucaine, levobupivacaine, lidocaine,
metabutethamine, metabutoxycaine, mepivacaine, meprylcaine,
propoxycaine, prilocaine, procaine, piperocaine, tetracaine,
trimecaine, and the like. The local injectable anesthetic compounds
can have multiple benefits in protein therapeutic formulations,
such as reduced viscosity, improved stability, and reduced pain
upon injection. In some embodiments, the local anesthetic compound
is present in the formulation in a concentration of about 50 mg/ml
or less.
[0055] In embodiments, a hindered amine having independent
pharmacological properties is used as an excipient compound in
accordance with the formulations and methods described herein. In
some embodiments, the excipient compounds possessing independent
pharmacological properties are present in an amount that does not
have a pharmacological effect and/or that is not therapeutically
effective. In other embodiments, the excipient compounds possessing
independent pharmacological properties are present in an amount
that does have a pharmacological effect and/or that is
therapeutically effective. In certain embodiments, a hindered amine
having independent pharmacological properties is used in
combination with another excipient compound that has been selected
to decrease formulation viscosity, where the hindered amine having
independent pharmacological properties is used to impart the
benefits of its pharmacological activity. For example, a local
injectable anesthetic compound can be used to decrease formulation
viscosity and also to reduce pain upon injection of the
formulation. The reduction of injection pain can be caused by
anesthetic properties; also a lower injection force can be required
when the viscosity is reduced by the excipients. Alternatively, a
local injectable anesthetic compound can be used to impart the
desirable pharmacological benefit of decreased local sensation
during formulation injection, while being combined with another
excipient compound that reduces the viscosity of the
formulation.
[0056] b. Excipient Compound Category 2: Anionic Aromatics
[0057] High concentration solutions of therapeutic or
non-therapeutic proteins can be formulated with anionic aromatic
small molecule compounds as excipient compounds. The anionic
aromatic excipient compounds can contain an aromatic functional
group such as phenyl, benzyl, aryl, alkylbenzyl, hydroxybenzyl,
phenolic, hydroxyaryl, heteroaromatic group, or a fused aromatic
group. The anionic aromatic excipient compounds also can contain an
anionic functional group such as carboxylate, oxide, phenoxide,
sulfonate, sulfate, phosphonate, phosphate, or sulfide. While the
anionic aromatic excipients might be described as an acid, a sodium
salt, or other, it is understood that the excipient can be used in
a variety of salt forms. Without being bound by theory, an anionic
aromatic excipient compound is thought to be a bulky, sterically
hindered molecule that can associate with cationic segments of a
protein, so that they can shield these sections of the protein,
thereby decreasing the interactions between protein molecules that
render the protein-containing formulation viscous.
[0058] In embodiments, examples of anionic aromatic excipient
compounds include compounds such as salicylic acid, aminosalicylic
acid, hydroxybenzoic acid, aminobenzoic acid, para-aminobenzoic
acid, benzenesulfonic acid, hydroxybenzenesulfonic acid,
naphthalenesulfonic acid, naphthalenedisulfonic acid, hydroquinone
sulfonic acid, sulfanilic acid, vanillic acid, vanillin,
vanillin-taurine adduct, aminophenol, anthranilic acid, cinnamic
acid, coumaric acid, adenosine monophosphate, indole acetic acid,
potassium urate, furan dicarboxylic acid, furan-2-acrylic acid,
2-furanpropionic acid, sodium phenylpyruvate, sodium
hydroxyphenylpyruvate, dihydroxybenzoic acid, trihydroxybenzoic
acid, pyrogallol, benzoic acid, and the salts of the foregoing
acids. In embodiments, the anionic aromatic excipient compounds is
formulated in the ionized salt form. In embodiments, an anionic
aromatic compound is formulated as the salt of a hindered amine,
such as dimethylcyclohexylammonium hydroxybenzoate. In embodiments,
the anionic aromatic excipient compounds is formulated with various
counterions such as organic cations. In embodiments, high
concentration solutions of therapeutic or non-therapeutic proteins
is formulated with anionic aromatic excipient compounds and
caffeine. In embodiments, the anionic aromatic excipient compounds
is metabolized in the body to yield biologically compatible
byproducts.
[0059] c. Excipient Compound Category 3: Functionalized Amino
Acids
[0060] High concentration solutions of therapeutic or
non-therapeutic proteins can be formulated with one or more
functionalized amino acids, where a single functionalized amino
acid or an oligopeptide comprising one or more functionalized amino
acids may be used as the excipient compound. In embodiments, the
functionalized amino acid compounds comprise molecules ("amino acid
precursors") that can be hydrolyzed or metabolized to yield amino
acids. In embodiments, the functionalized amino acids can contain
an aromatic functional group such as phenyl, benzyl, aryl,
alkylbenzyl, hydroxybenzyl, hydroxyaryl, heteroaromatic group, or a
fused aromatic group. In embodiments, the functionalized amino acid
compounds can contain esterified amino acids, such as methyl,
ethyl, propyl, butyl, benzyl, cycloalkyl, glyceryl, hydroxyethyl,
hydroxypropyl, PEG, and PPG esters. In embodiments, the
functionalized amino acid compounds are selected from the group
consisting of arginine ethyl ester, arginine methyl ester, arginine
hydroxyethyl ester, and arginine hydroxypropyl ester. In
embodiments, the functionalized amino acid compound is a charged
ionic compound in aqueous solution at neutral pH. For example, a
single amino acid can be derivatized by forming an ester, like an
acetate or a benzoate, and the hydrolysis products would be acetic
acid or benzoic acid, both natural materials, plus the amino acid.
In embodiments, the functionalized amino acid excipient compounds
is metabolized in the body to yield biologically compatible
byproducts.
[0061] d. Excipient Compound Category 4: Oligopeptides
[0062] High concentration solutions of therapeutic or
non-therapeutic proteins can be formulated with oligopeptides as
excipient compounds. In embodiments, the oligopeptide is designed
such that the structure has a charged section and a bulky section.
In embodiments, the oligopeptides consist of between 2 and 10
peptide subunits. The oligopeptide can be bi-functional, for
example a cationic amino acid coupled to a non-polar one, or an
anionic one coupled to a non-polar one. In embodiments, the
oligopeptides consist of between 2 and 5 peptide subunits. In
embodiments, the oligopeptides are homopeptides such as
polyglutamic acid, polyaspartic acid, poly-lysine, poly-arginine,
and poly-histidine. In embodiments, the oligopeptides have a net
cationic charge. In other embodiments, the oligopeptides are
heteropeptides, such as Trp2Lys3. In embodiments, the oligopeptide
can have an alternating structure such as an ABA repeating pattern.
In embodiments, the oligopeptide can contain both anionic and
cationic amino acids, for example, Arg-Glu. Without being bound by
theory, the oligopeptides comprise structures that can associate
with proteins in such a way that it reduces the intermolecular
interactions that lead to high viscosity solutions; for example,
the oligopeptide-protein association can be a charge-charge
interaction, leaving a somewhat non-polar amino acid to disrupt
hydrogen bonding of the hydration layer around the protein, thus
lowering viscosity. In some embodiments, the oligopeptide excipient
is present in the composition in a concentration of about 50 mg/ml
or less.
[0063] e. Excipient Compound Category 5: Short-Chain Organic
Acids
[0064] As used herein, the term "short-chain organic acids" refers
to C2-C6 organic acid compounds and the salts, esters, or lactones
thereof. This category includes saturated and unsaturated
carboxylic acids, hydroxy functionalized carboxylic acids, and
linear, branched, or cyclic carboxylic acids. In embodiments, the
acid group in the short-chain organic acid is a carboxylic acid,
sulfonic acid, phosphonic acid, or a salt thereof.
[0065] In addition to the four excipient categories above, high
concentration solutions of therapeutic or non-therapeutic proteins
can be formulated with short-chain organic acids, for example, the
acid or salt forms of sorbic acid, valeric acid, propionic acid,
caproic acid, and ascorbic acid as excipient compounds. Examples of
excipient compounds in this category include potassium sorbate,
taurine, calcium propionate, magnesium propionate, and sodium
ascorbate.
[0066] f. Excipient Compound Category 6: Low Molecular Weight
Aliphatic Polyacids
[0067] High concentration solutions of therapeutic or
non-therapeutic PEGylated proteins can be formulated with certain
excipient compounds that enable lower solution viscosity, where
such excipient compounds are low molecular weight aliphatic
polyacids. As used herein, the term "low molecular weight aliphatic
polyacids" refers to organic aliphatic polyacids having a molecular
weight <about 1500, and having at least two acidic groups, where
an acidic group is understood to be a proton-donating moiety.
Non-limiting examples of acidic groups include carboxylate,
phosphonate, phosphate, sulfonate, sulfate, nitrate, and nitrite
groups. Acidic groups on the low molecular weight aliphatic
polyacid can be in the anionic salt form such as carboxylate,
phosphonate, phosphate, sulfonate, sulfate, nitrate, and nitrite;
their counterions can be sodium, potassium, lithium, and ammonium.
Specific examples of low molecular weight aliphatic polyacids
useful for interacting with PEGylated proteins as described herein
include maleic acid, tartaric acid, glutaric acid, malonic acid,
citric acid, ethylenediaminetetraacetic acid (EDTA), aspartic acid,
glutamic acid, alendronic acid, etidronic acid and salts thereof.
Further examples of low molecular weight aliphatic polyacids in
their anionic salt form include phosphate (PO.sub.4.sup.3-),
hydrogen phosphate (HPO.sub.4.sup.3-), dihydrogen phosphate
(H.sub.2PO.sub.4.sup.-), sulfate (SO.sub.4.sup.2-), bisulfate
(HSO.sub.4.sup.-), pyrophosphate (P.sub.2O.sub.7.sup.4-), carbonate
(CO.sub.3.sup.2-), and bicarbonate (HCO.sub.3.sup.-). The
counterion for the anionic salts can be Na, Li, K, or ammonium ion.
These excipients can also be used in combination with excipients.
As used herein, the low molecular weight aliphatic polyacid can
also be an alpha hydroxy acid, where there is a hydroxyl group
adjacent to a first acidic group, for example glycolic acid, lactic
acid, and gluconic acid and salts thereof. In embodiments, the low
molecular weight aliphatic polyacid is an oligomeric form that
bears more than two acidic groups, for example polyacrylic acid,
polyphosphates, polypeptides and salts thereof. In some
embodiments, the low molecular weight aliphatic polyacid excipient
is present in the composition in a concentration of about 50 mg/ml
or less.
5. PROTEIN/EXCIPIENT SOLUTIONS: PROPERTIES AND PROCESSES
[0068] In certain embodiments, solutions of therapeutic or
non-therapeutic proteins is formulated with the above-identified
excipient compounds, such as hindered amines, anionic aromatics,
functionalized amino acids, oligopeptides, short-chain organic
acids to result in an improved protein-protein interaction
characteristics as measured by the protein diffusion interaction
parameter, kD, or the second virial coefficient, B22. As used
herein, an "improvement" in protein-protein interaction
characteristics achieved by formulations using the above-identified
excipient compounds means a decrease in protein-protein
interactions. These measurements of kD and B22 can be made using
standard techniques in the industry, and can be an indicator of
improved solution properties or stability of the protein in
solution. For example, a highly negative kD value can indicate that
the protein has a strong attractive interaction and this can lead
to aggregation, instability, and rheology problems. When formulated
in the presence of certain of the above identified excipient
compounds, the same protein can have a less negative kD value, or a
kD value near or above zero.
[0069] In embodiments, certain of the above-described excipient
compounds, such as hindered amines, anionic aromatics,
functionalized amino acids, oligopeptides, short-chain organic
acids, and/or low molecular weight aliphatic polyacids are used to
improve a protein-related process, such as the manufacture,
processing, sterile filling, purification, and analysis of
protein-containing solutions, using processing methods such as
filtration, syringing, transferring, pumping, mixing, heating or
cooling by heat transfer, gas transfer, centrifugation,
chromatography, membrane separation, centrifugal concentration,
tangential flow filtration, radial flow filtration, axial flow
filtration, lyophilization, and gel electrophoresis. These
processes and processing methods can have improved efficiency due
to the lower viscosity, improved solubility, or improved stability
of the proteins in the solution during manufacture, processing,
purification, and analysis steps. Additionally, equipment-related
processes such as the cleanup, sterilization, and maintenance of
protein processing equipment can be facilitated by the use of the
above-identified excipients due to decreased fouling, decreased
denaturing, lower viscosity, and improved solubility of the
protein.
[0070] High concentration solutions of therapeutic proteins
formulated with the above described excipient compounds can be
administered to patients using pre-filled syringes.
EXAMPLES
Materials
[0071] Bovine gamma globulin (BGG), >99% purity, Sigma Aldrich
[0072] Histidine, Sigma Aldrich [0073] Other materials described in
the examples below were from Sigma Aldrich unless otherwise
specified.
Example 1
Preparation of Formulations Containing Excipient Compounds and Test
Protein
[0074] Formulations were prepared using an excipient compound and a
test protein, where the test protein was intended to simulate
either a therapeutic protein that would be used in a therapeutic
formulation, or a non-therapeutic protein that would be used in a
non-therapeutic formulation. Such formulations were prepared in 50
mM histidine hydrochloride with different excipient compounds for
viscosity measurement in the following way. Histidine hydrochloride
was first prepared by dissolving 1.94 g histidine (Sigma-Aldrich,
St. Louis, Mo.) in distilled water and adjusting the pH to about
6.0 with 1 M hydrochloric acid (Sigma-Aldrich, St. Louis, Mo.) and
then diluting to a final volume of 250 mL with distilled water in a
volumetric flask. Excipient compounds were then dissolved in 50 mM
histidine HCl. Lists of excipients are provided below in Examples
4, 5, 6, and 7. In some cases excipient compounds were adjusted to
pH 6 prior to dissolving in 50 mM histidine HCl. In this case the
excipient compounds were first dissolved in deionized water at
about 5 wt % and the pH was adjusted to about 6.0 with either
hydrochloric acid or sodium hydroxide. The prepared salt solution
was then placed in a convection laboratory oven at about 150
degrees Fahrenheit (about 65 degrees C.) to evaporate the water and
isolate the solid excipient. Once excipient solutions in 50 mM
histidine HCl had been prepared, the test protein (bovine gamma
globulin (BGG) (Sigma-Aldrich, St. Louis, Mo.)) was dissolved at a
ratio of about 0.336 g BGG per 1 mL excipient solution. This
resulted in a final protein concentration of about 280 mg/mL.
Solutions of BGG in 50 mM histidine HCl with excipient were
formulated in 20 mL vials and allowed to shake at 100 rpm on an
orbital shaker table overnight. BGG solutions were then transferred
to 2 mL microcentrifuge tubes and centrifuged for ten minutes at
2300 rpm in an IEC MicroMax microcentrifuge to remove entrained air
prior to viscosity measurement.
Example 2
Viscosity Measurement
[0075] Viscosity measurements of formulations prepared as described
in Example 1 were made with a DV-IIT LV cone and plate viscometer
(Brookfield Engineering, Middleboro, Mass.). The viscometer was
equipped with a CP-40 cone and was operated at 3 rpm and 25 degrees
C. The formulation was loaded into the viscometer at a volume of
0.5 mL and allowed to incubate at the given shear rate and
temperature for 3 minutes, followed by a measurement collection
period of twenty seconds. This was then followed by 2 additional
steps consisting of 1 minute of shear incubation and subsequent
twenty-second measurement collection period. The three data points
collected were then averaged and recorded as the viscosity for the
sample.
Example 3
Protein Concentration Measurement
[0076] The concentration of the protein in the experimental
solutions was determined by measuring the absorbance of the protein
solution at a wavelength of 280 nm in a UV/VIS Spectrometer (Perkin
Elmer Lambda 35). First the instrument was calibrated to zero
absorbance with a 50 mM histidine buffer at pH 6. Next the protein
solutions were diluted by a factor of 300 with the same histidine
buffer and the absorbance at 280 nm recorded. The final
concentration of the protein in the solution was calculated by
using the extinction coefficient value of 1.264
mL/(mg.times.cm).
Example 4
Formulations with Hindered Amine Excipient Compounds
[0077] Formulations containing 280 mg/mL BGG were prepared as
described in Example 1, with some samples containing added
excipient compounds. In these tests, the hydrochloride salts of
dimethylcyclohexylamine (DMCHA), dicyclohexylmethylamine (DCHMA),
dimethylaminopropylamine (DMAPA), triethanolamine (TEA),
dimethylethanolamine (DMEA), and niacinamide were tested as
examples of the hindered amine excipient compounds. Also a
hydroxybenzoic acid salt of DMCHA and a taurine-dicyandiamide
adduct were tested as examples of the hindered amine excipient
compounds. The viscosity of each protein solution was measured as
described in Example 2, and the results are presented in Table 1
below, showing the benefit of the added excipient compounds in
reducing viscosity.
TABLE-US-00001 TABLE 1 Excipient Concen- Vis- Vis- Test tration
cosity cosity Number Excipient Added (mg/mL) (cP) Reduction 4.1
None 0 79 0% 4.2 DMCHA-HCl 28 50 37% 4.3 DMCHA-HCl 41 43 46% 4.4
DMCHA-HCl 50 45 43% 4.5 DMCHA-HCl 82 36 54% 4.6 DMCHA-HCl 123 35
56% 4.7 DMCHA-HCl 164 40 49% 4.8 DMAPA-HCl 87 57 28% 4.9 DMAPA-HCl
40 54 32% 4.10 DCHMA-HCl 29 51 35% 4.11 DCHMA-HCl 50 51 35% 4.14
TEA-HCl 97 51 35% 4.15 TEA-HCl 38 57 28% 4.16 DMEA-HCl 51 51 35%
4.17 DMEA-HCl 98 47 41% 4.20 DMCHA-hydroxybenzoate 67 46 42% 4.21
DMCHA-hydroxybenzoate 92 42 47% 4.22 Product of Example 8 26 58 27%
4.23 Product of Example 8 58 50 37% 4.24 Product of Example 8 76 49
38% 4.25 Product of Example 8 103 46 42% 4.26 Product of Example 8
129 47 41% 4.27 Product of Example 8 159 42 47% 4.28 Product of
Example 8 163 42 47% 4.29 Niacinamide 48 39 51% 4.30
N-Methyl-2-pyrrolidone 30 45 43% 4.31 N-Methyl-2-pyrrolidone 52 52
34%
Example 5
Formulations with Anionic Aromatic Excipient Compounds
[0078] Formulations of 280 mg/mL BGG were prepared as described in
Example 1, with some samples containing added excipient compounds.
The viscosity of each solution was measured as described in Example
2, and the results are presented in Table 2 below, showing the
benefit of the added excipient compounds in reducing viscosity.
TABLE-US-00002 TABLE 2 Excipient Concen- Vis- Vis- Test tration
cosity cosity Number Excipient Added (mg/mL) (cP) Reduction 5.1
None 0 79 0% 5.2 Sodium aminobenzoate 43 48 39% 5.3 Sodium
hydroxybenzoate 26 50 37% 5.4 Sodium sulfanilate 44 49 38% 5.5
Sodium sulfanilate 96 42 47% 5.6 Sodium indole acetate 52 58 27%
5.7 Sodium indole acetate 27 78 1% 5.8 Vanillic acid, sodium salt
25 56 29% 5.9 Vanillic acid, sodium salt 50 50 37% 5.10 Sodium
salicylate 25 57 28% 5.11 Sodium salicylate 50 52 34% 5.12
Adenosine monophosphate 26 47 41% 5.13 Adenosine monophosphate 50
66 16% 5.14 Sodium benzoate 31 61 23% 5.15 Sodium benzoate 56 62
22%
Example 6
Formulations with Oligopeptide Excipient Compounds
[0079] Oligopeptides (n=5) were synthesized by NeoBioLab Inc. in
>95% purity with the N terminus as a free amine and the C
terminus as a free acid. Dipeptides (n=2) were synthesized by
LifeTein LLC in 95% purity. Formulations of 280 mg/mL BGG were
prepared as described in Example 1, with some samples containing
the synthetic oligopeptides as added excipient compounds. The
viscosity of each solution was measured as described in Example 2,
and the results are presented in Table 3 below, showing the benefit
of the added excipient compounds in reducing viscosity.
TABLE-US-00003 TABLE 3 Excipient Concen- Vis- Vis- Test tration
cosity cosity Number Excipient Added (mg/mL) (cP) Reduction 6.1
None 0 79 0% 6.2 ArgX5 100 55 30% 6.3 ArgX5 50 54 32% 6.4 HisX5 100
62 22% 6.5 HisX5 50 51 35% 6.6 HisX5 25 60 24% 6.7 Trp2Lys3 100 59
25% 6.8 Trp2Lys3 50 60 24% 6.9 AspX5 100 102 -29% 6.10 AspX5 50 82
-4% 6.11 Dipeptide LE (Leu-Glu) 50 72 9% 6.12 Dipeptide YE
(Tyr-Glu) 50 55 30% 6.13 Dipeptide RP (Arg-Pro) 50 51 35% 6.14
Dipeptide RK (Arg-Lys) 50 53 33% 6.15 Dipeptide RH (Arg-His) 50 52
34% 6.16 Dipeptide RR (Arg-Arg) 50 57 28% 6.17 Dipeptide RE
(Arg-Glu) 50 50 37% 6.18 Dipeptide LE (Leu-Glu) 100 87 -10% 6.19
Dipeptide YE (Tyr-Glu) 100 68 14% 6.20 Dipeptide RP (Arg-Pro) 100
53 33% 6.21 Dipeptide RK (Arg-Lys) 100 64 19% 6.22 Dipeptide RH
(Arg-His) 100 72 9% 6.23 Dipeptide RR (Arg-Arg) 100 62 22% 6.24
Dipeptide RE (Arg-Glu) 100 66 16%
Example 8
Synthesis of Guanyl Taurine Excipient
[0080] Guanyl taurine was prepared following method described in
U.S. Pat. No. 2,230,965. Taurine (Sigma-Aldrich, St. Louis, Mo.)
3.53 parts were mixed with 1.42 parts of dicyandiamide
(Sigma-Aldrich, St. Louis, Mo.) and grinded in a mortar and pestle
until a homogeneous mixture was obtained. Next the mixture was
placed in a flask and heated at 200.degree. C. for 4 hours. The
product was used without further purification.
Example 9
Protein Formulations Containing Excipient Compounds
[0081] Formulations were prepared using an excipient compound and a
test protein, where the test protein was intended to simulate
either a therapeutic protein that would be used in a therapeutic
formulation, or a non-therapeutic protein that would be used in a
non-therapeutic formulation. Such formulations were prepared in 50
mM aqueous histidine hydrochloride buffer solution with different
excipient compounds for viscosity measurement in the following way.
Histidine hydrochloride buffer solution was first prepared by
dissolving 1.94 g histidine (Sigma-Aldrich, St. Louis, Mo.) in
distilled water and adjusting the pH to about 6.0 with 1 M
hydrochloric acid (Sigma-Aldrich, St. Louis, Mo.) and then diluting
to a final volume of 250 mL with distilled water in a volumetric
flask. Excipient compounds were then dissolved in the 50 mM
histidine HCl buffer solution. A list of the excipient compounds is
provided in Table 4. In some cases excipient compounds were
dissolved in 50 mM histidine HCl and the resulting solution pH was
adjusted with small amounts of concentrated sodium hydroxide or
hydrochloric acid to achieve pH 6 prior to dissolution of the model
protein. In some cases excipient compounds were adjusted to pH 6
prior to dissolving in 50 mM histidine HCl. In this case the
excipient compounds were first dissolved in deionized water at
about 5 wt % and the pH was adjusted to about 6.0 with either
hydrochloric acid or sodium hydroxide. The prepared salt solution
was then placed in a convection laboratory oven at about 150
degrees Fahrenheit (65 degrees C.) to evaporate the water and
isolate the solid excipient. Once excipient solutions in 50 mM
histidine HCl had been prepared, the test protein, bovine gamma
globulin (Sigma-Aldrich, St. Louis, Mo.) was dissolved at a ratio
to achieve a final protein concentration of about 280 mg/mL.
Solutions of BGG in 50 mM histidine HCl with excipient were
formulated in 20 mL vials and allowed to shake at 100 rpm on an
orbital shaker table overnight. BGG solutions were then transferred
to 2 mL microcentrifuge tubes and centrifuged for ten minutes at
2300 rpm in an IEC MicroMax microcentrifuge to remove entrained air
prior to viscosity measurement.
[0082] Viscosity measurements of formulations prepared as described
above were made with a DV-IIT LV cone and plate viscometer
(Brookfield Engineering, Middleboro, Mass.). The viscometer was
equipped with a CP-40 cone and was operated at 3 rpm and 25 degrees
C. The formulation was loaded into the viscometer at a volume of
0.5 mL and allowed to incubate at the given shear rate and
temperature for 3 minutes, followed by a measurement collection
period of twenty seconds. This was then followed by 2 additional
steps consisting of 1 minute of shear incubation and subsequent
twenty-second measurement collection period. The three data points
collected were then averaged and recorded as the viscosity for the
sample. Viscosities of solutions with excipient were normalized to
the viscosity of the model protein solution without excipient. The
normalized viscosity is the ratio of the viscosity of the model
protein solution with excipient to the viscosity of the model
protein solution with no excipient.
TABLE-US-00004 TABLE 4 Excipient Normalized Concen- Vis- Vis- Test
tration cosity cosity Number Excipient Added (mg/mL) (cP) Reduction
9.1 DMCHA-HCl 120 0.44 56% 9.2 Niacinamide 50 0.51 49% 9.3
Isonicotinamide 50 0.48 52% 9.4 Tyramine HCl 70 0.41 59% 9.5
Histamine HCl 50 0.41 59% 9.6 Imidazole HCl 100 0.43 57% 9.7
2-methyl-2- 60 0.43 57% imidazoline HCl 9.8 1-butyl-3-methyl- 100
0.48 52% imidazolium chloride 9.9 Procaine HCl 50 0.53 47% 9.10
3-aminopyridine 50 0.51 49% 9.11 2,4,6-trimethyl- 50 0.49 51%
pyridine 9.12 3-pyridine methanol 50 0.53 47% 9.13 Nicotinamide
adenine 20 0.56 44% dinucleotide 9.15 Sodium phenylpyruvate 55 0.57
43% 9.16 2-Pyrrolidinone 60 0.68 32% 9.17 Morpholine HCl 50 0.60
40% 9.18 Agmatine sulfate 55 0.77 23% 9.19 1-butyl-3-methyl- 60
0.66 34% imidazolium iodide 9.21 L-Anserine nitrate 50 0.79 21%
9.22 1-hexyl-3-methyl- 65 0.89 11% imidazolium chloride 9.23
N,N-diethyl 50 0.67 33% nicotinamide 9.24 Nicotinic acid, 100 0.54
46% sodium salt 9.25 Biotin 20 0.69 31%
Example 10
Preparation of Formulations Containing Excipient Combinations and
Test Protein
[0083] Formulations were prepared using a primary excipient
compound, a secondary excipient compound and a test protein, where
the test protein was intended to simulate either a therapeutic
protein that would be used in a therapeutic formulation, or a
non-therapeutic protein that would be used in a non-therapeutic
formulation. The primary excipient compounds were selected from
compounds having both anionic and aromatic functionality, as listed
below in Table 5. The secondary excipient compounds were selected
from compounds having either nonionic or cationic charge at pH 6
and either imidazoline or benzene rings, as listed below in Table
5. Formulations of these excipients were prepared in 50 mM
histidine hydrochloride buffer solution for viscosity measurement
in the following way. Histidine hydrochloride was first prepared by
dissolving 1.94 g histidine (Sigma-Aldrich, St. Louis, Mo.) in
distilled water and adjusting the pH to about 6.0 with 1 M
hydrochloric acid (Sigma-Aldrich, St. Louis, Mo.) and then diluting
to a final volume of 250 mL with distilled water in a volumetric
flask. The individual primary or secondary excipient compounds were
then dissolved in 50 mM histidine HCl. Combinations of primary and
secondary excipients were dissolved in 50 mM histidine HCl and the
resulting solution pH adjusted with small amounts of concentrated
sodium hydroxide or hydrochloric acid to achieve pH 6 prior to
dissolution of the model protein. Once excipient solutions had been
prepared as described above, the test protein (bovine gamma
globulin (BGG) (Sigma-Aldrich, St. Louis, Mo.) was dissolved into
each test solution at a ratio to achieve a final protein
concentration of about 280 mg/mL. Solutions of BGG in 50 mM
histidine HCl with excipient were formulated in 20 mL vials and
allowed to shake at 100 rpm on an orbital shaker table overnight.
BGG solutions were then transferred to 2 mL microcentrifuge tubes
and centrifuged for ten minutes at 2300 rpm in an IEC MicroMax
microcentrifuge to remove entrained air prior to viscosity
measurement.
[0084] Viscosity measurements of formulations prepared as described
above were made with a DV-IIT LV cone and plate viscometer
(Brookfield Engineering, Middleboro, Mass.). The viscometer was
equipped with a CP-40 cone and was operated at 3 rpm and 25 degrees
C. The formulation was loaded into the viscometer at a volume of
0.5 mL and allowed to incubate at the given shear rate and
temperature for 3 minutes, followed by a measurement collection
period of twenty seconds. This was then followed by 2 additional
steps consisting of 1 minute of shear incubation and a subsequent
twenty-second measurement collection period. The three data points
collected were then averaged and recorded as the viscosity for the
sample. Viscosities of solutions with excipient were normalized to
the viscosity of the model protein solution without excipient, and
summarized in Table 5 below. The normalized viscosity is the ratio
of the viscosity of the model protein solution with excipient to
the viscosity of the model protein solution with no excipient. The
example shows that a combination of primary and secondary
excipients can give a better result than a single excipient.
TABLE-US-00005 TABLE 5 Primary Excipient Secondary Excipient
Concen- Concen- Normalized Test tration tration Vis- Number Name
(mg/mL) Name (mg/mL) cosity 10.1 Salicylic 30 None 0 0.79 Acid 10.2
Salicylic 25 Imidazole 4 0.59 Acid 10.3 4-hydroxy- 30 None 0 0.61
benzoic acid 10.4 4-hydroxy- 25 Imidazole 5 0.57 benzoic acid 10.5
4-hydroxy- 31 None 0 0.59 benzene sulfonic acid 10.6 4-hydroxy- 26
Imidazole 5 0.70 benzene sulfonic acid 10.7 4-hydroxy- 25 Caffeine
5 0.69 benzene sulfonic acid 10.8 None 0 Caffeine 10 0.73 10.9 None
0 Imidazole 5 0.75
Example 11
Preparation of Formulations Containing Excipient Combinations and
Test Protein
[0085] Formulations were prepared using a primary excipient
compound, a secondary excipient compound and a test protein, where
the test protein was intended to simulate a therapeutic protein
that would be used in a therapeutic formulation, or a
non-therapeutic protein that would be used in a non-therapeutic
formulation. The primary excipient compounds were selected from
compounds having both anionic and aromatic functionality, as listed
below in Table 6. The secondary excipient compounds were selected
from compounds having either nonionic or cationic charge at pH 6
and either imidazoline or benzene rings, as listed below in Table
6. Formulations of these excipients were prepared in distilled
water for viscosity measurement in the following way. Combinations
of primary and secondary excipients were dissolved in distilled
water and the resulting solution pH adjusted with small amounts of
concentrated sodium hydroxide or hydrochloric acid to achieve pH 6
prior to dissolution of the model protein. Once excipient solutions
in distilled water had been prepared, the test protein (bovine
gamma globulin (BGG) (Sigma-Aldrich, St. Louis, Mo.)) was dissolved
at a ratio to achieve a final protein concentration of about 280
mg/mL. Solutions of BGG in distilled water with excipient were
formulated in 20 mL vials and allowed to shake at 100 rpm on an
orbital shaker table overnight. BGG solutions were then transferred
to 2 mL microcentrifuge tubes and centrifuged for ten minutes at
2300 rpm in an IEC MicroMax microcentrifuge to remove entrained air
prior to viscosity measurement.
[0086] Viscosity measurements of formulations prepared as described
above were made with a DV-IIT LV cone and plate viscometer
(Brookfield Engineering, Middleboro, Mass.). The viscometer was
equipped with a CP-40 cone and was operated at 3 rpm and 25 degrees
C. The formulation was loaded into the viscometer at a volume of
0.5 mL and allowed to incubate at the given shear rate and
temperature for 3 minutes, followed by a measurement collection
period of twenty seconds. This was then followed by 2 additional
steps consisting of 1 minute of shear incubation and a subsequent
twenty-second measurement collection period. The three data points
collected were then averaged and recorded as the viscosity for the
sample. Viscosities of solutions with excipient were normalized to
the viscosity of the model protein solution without excipient, and
summarized in Table 6 below. The normalized viscosity is the ratio
of the viscosity of the model protein solution with excipient to
the viscosity of the model protein solution with no excipient. The
example shows that a combination of primary and secondary
excipients can give a better result than a single excipient.
TABLE-US-00006 TABLE 6 Primary Excipient Secondary Excipient
Concen- Concen- Normalized Test tration tration Vis- Number Name
(mg/mL) Name (mg/mL) cosity 11.1 Salicylic 20 None 0 0.96 Acid 11.2
Salicylic 20 Caffeine 5 0.71 Acid 11.3 Salicylic 20 Niacinamide 5
0.76 Acid 11.4 Salicylic 20 Imidazole 5 0.73 Acid
Example 12
Preparation of Formulations Containing Excipient Compounds and
PEG
[0087] Materials: All materials were purchased from Sigma-Aldrich,
St. Louis, Mo. Formulations were prepared using an excipient
compound and PEG, where the PEG was intended to simulate a
therapeutic PEGylated protein that would be used in a therapeutic
formulation. Such formulations were prepared by mixing equal
volumes of a solution of PEG with a solution of the excipient. Both
solutions were prepared in a Tris buffer consisting of 10 mM Tris,
135 mM NaCl, 1 mM trans-cinnamic acid at pH of 7.3.
[0088] The PEG solution was prepared by mixing 3 g of Poly(ethylene
oxide) average Mw .about.1,000,000 (Aldrich Catalog #372781) with
97 g of the Tris buffer solution. The mixture was stirred overnight
for complete dissolution.
[0089] An example of the excipient solution preparation is as
follows: An approximately 80 mg/mL solution of citric acid in the
Tris buffer was prepared by dissolving 0.4 g of citric acid
(Aldrich cat. #251275) in 5 mL of the Tris buffer solution and
adjusted the pH to 7.3 with minimum amount of 10 M NaOH
solution.
[0090] The PEG excipient solution was prepared by mixing 0.5 mL of
the PEG solution with 0.5 mL of the excipient solution and mixed by
using a vortex for a few seconds. A control sample was prepared by
mixing 0.5 mL of the PEG solution with 0.5 mL of the Tris buffer
solution.
Example 13
Viscosity Measurements of Formulations Containing Excipient
Compounds and PEG
[0091] Viscosity measurements of the formulations prepared were
made with a DV-IIT LV cone and plate viscometer (Brookfield
Engineering, Middleboro, Mass.). The viscometer was equipped with a
CP-40 cone and was operated at 3 rpm and 25 degrees C. The
formulation was loaded into the viscometer at a volume of 0.5 mL
and allowed to incubate at the given shear rate and temperature for
3 minutes, followed by a measurement collection period of twenty
seconds. This was then followed by 2 additional steps consisting of
1 minute of shear incubation and subsequent twenty second
measurement collection period. The three data points collected were
then averaged and recorded as the viscosity for the sample.
[0092] The results presented in Table 7 show the effect of the
added excipient compounds in reducing viscosity.
TABLE-US-00007 TABLE 7 Excipient Concen- Vis- Vis- Test tration
cosity cosity Number Excipient (mg/mL) (cP) Reduction 13.1 None 0
104.8 0% 13.2 Citric acid Na salt 40 56.8 44% 13.3 Citric acid Na
salt 20 73.3 28% 13.4 glycerol phosphate 40 71.7 30% 13.5 glycerol
phosphate 20 83.9 18% 13.6 Ethylene diamine 40 84.7 17% 13.7
Ethylene diamine 20 83.9 15% 13.8 EDTA/K salt 40 67.1 36% 13.9
EDTA/K salt 20 76.9 27% 13.10 EDTA/Na salt 40 68.1 35% 13.11
EDTA/Na salt 20 77.4 26% 13.12 D-Gluconic acid/K salt 40 80.32 23%
13.13 D-Gluconic acid/K salt 20 88.4 16% 13.14 D-Gluconic acid/Na
salt 40 81.24 23% 13.15 D-Gluconic acid/Na salt 20 86.6 17% 13.16
lactic acid/K salt 40 80.42 23% 13.17 lactic acid/K salt 85.1 19%
13.18 lactic acid/Na salt 40 86.55 17% 13.19 lactic acid/Na salt 20
87.2 17% 13.20 etidronic acid/K salt 24 71.91 31% 13.21 etidronic
acid/K salt 12 80.5 23% 13.22 etidronic acid/Na salt 24 71.6 32%
13.23 etidronic acid/Na salt 12 79.4 24%
Example 14
Preparation of PEGylated BSA with 1 PEG Chain Per BSA Molecule
[0093] To a beaker was added 200 mL of a phosphate buffered saline
(Aldrich Cat. # P4417) and 4 g of BSA (Aldrich Cat. # A7906) and
mixed with a magnetic bar. Next 400 mg of methoxy polyethylene
glycol maleimide, MW=5,000, (Aldrich Cat. #63187) was added. The
reaction mixture was allowed to react overnight at room
temperature. The following day, 20 drops of HCl 0.1 M were added to
stop the reaction. The reaction product was characterized by
SDS-Page and SEC which clearly showed the PEGylated BSA. The
reaction mixture was placed in an Amicon centrifuge tube with a
molecular weight cutoff (MWCO) of 30,000 and concentrated to a few
milliliters. Next the sample was diluted 20 times with a histidine
buffer, 50 mM at a pH of approximately 6, followed by concentrating
until a high viscosity fluid was obtained. The final concentration
of the protein solution was obtained by measuring the absorbance at
280 nm and using a coefficient of extinction for the BSA of 0.6678.
The results indicated that the final concentration of BSA in the
solution was 342 mg/mL.
Example 15
Preparation of PEGylated BSA with Multiple PEG Chains Per BSA
Molecule
[0094] A 5 mg/mL solution of BSA (Aldrich A7906) in phosphate
buffer, 25 mM at pH of 7.2, was prepared by mixing 0.5 g of the BSA
with 100 mL of the buffer. Next 1 g of a methoxy PEG
propionaldehyde Mw=20,000 (JenKem Technology, Plano, Tex. 75024)
was added followed by 0.12 g of sodium cyanoborohydride (Aldrich
156159). The reaction was allowed to proceed overnight at room
temperature. The following day the reaction mixture was diluted 13
times with a Tris buffer (10 mM Tris, 135 mM NaCl at pH=7.3) and
concentrated using Amicon centrifuge tubes MWCO of 30,000 until a
concentration of approximately 150 mg/mL was reached.
Example 16
Preparation of PEGylated Lysozyme with Multiple PEG Chains Per
Lysozyme Molecule
[0095] A 5 mg/mL solution of lysozyme (Aldrich L6876) in phosphate
buffer, 25 mM at pH of 7.2, was prepared by mixing 0.5 g of the
lysozyme with 100 mL of the buffer. Next 1 g of a methoxy PEG
propionaldehyde Mw=5,000 (JenKem Technology, Plano, Tex. 75024) was
added followed by 0.12 g of Sodium cyanoborohydride (Aldrich
156159). The reaction was allowed to proceed overnight at room
temperature. The following day the reaction mixture was diluted 49
times with the phosphate buffer, 25 mM at pH of 7.2, and
concentrated using Amicon centrifuge tubes MWCO of 30,000. The
final concentration of the protein solution was obtained by
measuring the absorbance at 280 nm and using a coefficient of
extinction for the lysozyme of 2.63. The final concentration of
lysozyme in the solution was 140 mg/mL.
Example 17
Effect of Excipients on Viscosity of PEGylated BSA with 1 PEG Chain
Per BSA Molecule
[0096] Formulations of PEGylated BSA (from Example 14 above) with
excipients were prepared by adding 6 or 12 milligrams of the
excipient salt to 0.3 mL of the PEGylated BSA solution. The
solution was mixed by gently shaking and the viscosity was measured
by a RheoSense microVisc equipped with an A10 channel (100 micron
depth) at a shear rate of 500 sec-1. The viscometer measurements
were completed at ambient temperature.
[0097] The results presented in Table 8 shows the effect of the
added excipient compounds in reducing viscosity.
TABLE-US-00008 TABLE 8 Excipient Concen- Vis- Vis- Test tration
cosity cosity Number Excipient (mg/mL) (cP) Reduction 17.1 None 0
228.6 0% 17.2 Alpha-Cyclodextrin 20 151.5 34% sulfated Na salt 17.3
K acetate 40 89.5 60%
Example 18
Effect of Excipients on Viscosity of PEGylated BSA with Multiple
PEG Chains Per BSA Molecule
[0098] A formulations of PEGylated BSA (from Example 15 above) with
citric acid Na salt as excipient was prepared by adding 8
milligrams of the excipient salt to 0.2 mL of the PEGylated BSA
solution. The solution was mixed by gently shaking and the
viscosity was measured by a RheoSense microVisc equipped with an
A10 channel (100 micron depth) at a shear rate of 500 sec-1. The
viscometer measurements were completed at ambient temperature. The
results presented in Table 9 shows the effect of the added
excipient compounds in reducing viscosity.
TABLE-US-00009 TABLE 9 Excipient Concen- Vis- Vis- Test tration
cosity cosity Number Excipient Added (mg/mL) (cP) Reduction 18.1
None 0 56.8 0% 18.2 Citric acid Na salt 40 43.5 23%
Example 19
Effect of Excipients on Viscosity of PEGylated Lysozyme with
Multiple PEG Chains Per Lysozyme Molecule
[0099] A formulation of PEGylated lysozyme (from Example 16 above)
with potassium acetate as excipient was prepared by adding 6
milligrams of the excipient salt to 0.3 mL of the PEGylated
lysozyme solution. The solution was mixed by gently shaking and the
viscosity was measured by a RheoSense microVisc equipped with an
A10 channel (100 micron depth) at a shear rate of 500 sec-1. The
viscometer measurements were completed at ambient temperature. The
results presented in the next table shows the benefit of the added
excipient compounds in reducing viscosity.
TABLE-US-00010 TABLE 10 Excipient Concen- Vis- Vis- Test tration
cosity cosity Number Excipient (mg/mL) (cP) Reduction 19.1 None 0
24.6 0% 19.2 K acetate 20 22.6 8%
Example 20
Protein Formulations Containing Excipient Combinations
[0100] Formulations were prepared using an excipient compound or a
combination of two excipient compounds and a test protein, where
the test protein was intended to simulate a therapeutic protein
that would be used in a therapeutic formulation. These formulations
were prepared in 20 mM histidine buffer with different excipient
compounds for viscosity measurement in the following way. Excipient
combinations were dissolved in 20 mM histidine (Sigma-Aldrich, St.
Louis, Mo.) and the resulting solution pH adjusted with small
amounts of concentrated sodium hydroxide or hydrochloric acid to
achieve pH 6 prior to dissolution of the model protein. Excipient
compounds for this Example are listed below in Table 11. Once
excipient solutions had been prepared, the test protein (bovine
gamma globulin or "BGG" (Sigma-Aldrich, St. Louis, Mo.)) was
dissolved at a ratio to achieve a final protein concentration of
about 280 mg/mL. Solutions of BGG in the excipient solutions were
formulated in 5 mL sterile polypropylene tubes and allowed to shake
at 80-100 rpm on an orbital shaker table overnight. BGG solutions
were then transferred to 2 mL microcentrifuge tubes and centrifuged
for about ten minutes at 2300 rpm in an IEC MicroMax
microcentrifuge to remove entrained air prior to viscosity
measurement.
[0101] Viscosity measurements of formulations prepared as described
above were made with a DV-IIT LV cone and plate viscometer
(Brookfield Engineering, Middleboro, Mass.). The viscometer was
equipped with a CP-40 cone and was operated at 3 rpm and 25 degrees
Centigrade. The formulation was loaded into the viscometer at a
volume of 0.5 mL and allowed to incubate at the given shear rate
and temperature for 3 minutes, followed by a measurement collection
period of twenty seconds. This was then followed by 2 additional
steps consisting of 1 minute of shear incubation and subsequent
twenty second measurement collection period. The three data points
collected were then averaged and recorded as the viscosity for the
sample. Viscosities of solutions with excipient were normalized to
the viscosity of the model protein solution without excipient, and
the results are shown in Table 11 below. The normalized viscosity
is the ratio of the viscosity of the model protein solution with
excipient to the viscosity of the model protein solution with no
excipient.
TABLE-US-00011 TABLE 11 Excipient A Excipient B Normalized Test
Conc. Conc. Vis- # Name (mg/mL) Name (mg/mL) cosity 20.1 None 0
None 0 1.00 20.2 Aspartame 10 None 0 0.83 Saccharin 60 None 0 0.51
20.4 Acesulfame K 80 None 0 0.44 20.5 Theophylline 10 None 0 0.84
20.6 Saccharin 30 None 0 0.58 20.7 Acesulfame K 40 None 0 0.61 20.8
Caffeine 15 Taurine 15 0.82 20.9 Caffeine 15 Tyramine 15 0.67
Example 21
Protein Formulations Containing Excipients to Reduce Viscosity and
Injection Pain
[0102] Formulations were prepared using an excipient compound, a
second excipient compound, and a test protein, where the test
protein was intended to simulate a therapeutic protein that would
be used in a therapeutic formulation. The first excipient compound,
Excipient A, was selected from a group of compounds having local
anesthetic properties. The first excipient, Excipient A and the
second excipient, Excipient B are listed in Table 12. These
formulations were prepared in 20 mM histidine buffer using
Excipient A and Excipient B in the following way, so that their
viscosities could be measured. Excipients in the amounts disclosed
in Table 12 were dissolved in 20 mM histidine (Sigma-Aldrich, St
Louis, Mo.) and the resulting solutions were pH adjusted with small
amounts of concentrated sodium hydroxide or hydrochloric acid to
achieve pH 6 prior to dissolution of the model protein. Once
excipient solutions had been prepared, the test protein (bovine
gamma globulin ("BGG") (Sigma-Aldrich, St. Louis, Mo.)) was
dissolved in the excipient solution at a ratio to achieve a final
protein concentration of about 280 mg/mL. Solutions of BGG in the
excipient solutions were formulated in 5 mL sterile polypropylene
tubes and allowed to shake at 80-100 rpm on an orbital shaker table
overnight. BGG-excipient solutions were then transferred to 2 mL
microcentrifuge tubes and centrifuged for about ten minutes at 2300
rpm in an IEC MicroMax microcentrifuge to remove entrained air
prior to viscosity measurement.
[0103] Viscosity measurements of the formulations prepared as
described above were made with a DV-IIT LV cone and plate
viscometer (Brookfield Engineering, Middleboro, Mass.). The
viscometer was equipped with a CP-40 cone and was operated at 3 rpm
and 25 degrees Centigrade. The formulation was loaded into the
viscometer at a volume of 0.5 mL and allowed to incubate at the
given shear rate and temperature for 3 minutes, followed by a
measurement collection period of twenty seconds. This was then
followed by 2 additional steps consisting of 1 minute of shear
incubation and subsequent twenty second measurement collection
period. The three data points collected were then averaged and
recorded as the viscosity for the sample. Viscosities of solutions
with excipient were normalized to the viscosity of the model
protein solution without excipient, and the results are shown in
Table 12 below. The normalized viscosity is the ratio of the
viscosity of the model protein solution with excipient to the
viscosity of the model protein solution with no excipient.
TABLE-US-00012 TABLE 12 Excipient A Excipient B Normalized Test
Conc. Conc. Vis- # Name (mg/mL) Name (mg/mL) cosity 21.1 None 0
None 0 1.00 21.2 Lidocaine 45 None 0 0.73 21.3 Lidocaine 23 None 0
0.74 21.4 Lidocaine 10 Caffeine 15 0.71 21.5 Procaine HCl 40 None 0
0.64 21.6 Procaine HCl 20 Caffeine 15 0.69
Example 22
Formulations Containing Excipient Compounds and PEG
[0104] Formulations were prepared using an excipient compound and
PEG, where the PEG was intended to simulate a therapeutic PEGylated
protein that would be used in a therapeutic formulation, and where
the excipient compounds were provided in the amounts as listed in
Table 13. These formulations were prepared by mixing equal volumes
of a solution of PEG with a solution of the excipient. Both
solutions were prepared in DI-Water.
[0105] The PEG solution was prepared by mixing 16.5 g of
poly(ethylene oxide) average Mw .about.100,000 (Aldrich Catalog
#181986) with 83.5 g of DI water. The mixture was stirred overnight
for complete dissolution.
[0106] The excipient solutions were prepared by this general method
and as detailed in Table 13 below: An approximately 20 mg/mL
solution of potassium phosphate tribasic (Aldrich Catalog # P5629)
in DI-water was prepared by dissolving 0.05 g of potassium
phosphate in 5 mL of DI-water. The PEG excipient solution was
prepared by mixing 0.5 mL of the PEG solution with 0.5 mL of the
excipient solution and mixed by using a vortex for a few seconds. A
control sample was prepared by mixing 0.5 mL of the PEG solution
with 0.5 mL of DI-water. Viscosity was measured and results are
recorded in Table 13 below.
TABLE-US-00013 TABLE 13 Excipient Vis- Concen- Vis- cosity Test
tration cosity Reduction Number Excipient (mg/mL) (cP) (%) 22.1
None 0 79.7 0 22.2 Citric acid Na salt 10 74.9 6.0 22.3 Potassium
phosphate 10 72.3 9.3 22.4 Citric acid Na salt/ 10/10 69.1 13.3
Potassium phosphate 22.5 Sodium sulfate 10 75.1 5.8 22.6 Citric
acid Na salt/ 10/10 70.4 11.7 Sodium sulfate
Example 23
Improved Processing of Protein Solutions with Excipients
[0107] Two BGG solutions were prepared by mixing 0.25 g of solid
BGG (Aldrich catalogue number G5009) with 4 ml of a buffer
solution. For Sample A: Buffer solution was 20 mM histidine buffer
(pH=6.0). For sample B: Buffer solution was 20 mM histidine buffer
containing 15 mg/ml of caffeine (pH=6). The dissolution of the
solid BGG was carried out by placing the samples in an orbital
shaker set at 100 rpm. The buffer sample containing caffeine
excipient was observed to dissolve the protein faster. For the
sample with the caffeine excipient (Sample B) complete dissolution
of the BGG was achieved in 15 minutes. For the sample without the
caffeine (Sample A) the dissolution needed 35 minutes.
[0108] Next the samples were placed in 2 separate Amicon Ultra 4
Centrifugal Filter Unit with a 30,000 molecular weight cut off and
the samples were centrifuged at 2,500 rpm at 10 minutes intervals.
The filtrate volume recovered after each 10 minute centrifuge run
was recorded. The results in Table 14 show the faster recovery of
the filtrate for Sample B. In addition Sample B kept concentrating
with every additional run but Sample A reached a maximum
concentration point and further centrifugation did not result in
further sample concentration.
TABLE-US-00014 TABLE 14 Centrifuge Sample A filtrate Sample B
filtrate time (min) collected (mL) collected (mL) 10 0.28 0.28 20
0.56 0.61 30 0.78 0.88 40 0.99 1.09 50 1.27 1.42 60 1.51 1.71 70
1.64 1.99 80 1.79 2.29 90 1.79 2.39 100 1.79 2.49
Example 24
Protein Formulations Containing Multiple Excipients
[0109] This example shows how the combination of caffeine and
arginine as excipients has a beneficial effect on decreasing
viscosity of a BGG solution. Four BGG solutions were prepared by
mixing 0.18 g of solid BGG (Aldrich catalogue number G5009) with
0.5 mL of a 20 mM Histidine buffer at pH 6. Each buffer solution
contained different excipient or combination of excipients as
described in the table below. The viscosity of the solutions was
measured as described in previous examples. The results show that
the hindered amine excipient, caffeine, can be combined with known
excipients such as arginine, and the combination has better
viscosity reduction properties than the individual excipients by
themselves.
TABLE-US-00015 TABLE 15 Vis- Vis- cosity cosity Reduction Sample
Excipient added (cP) (%) A None 130.6 0 B Caffeine (10 mg/ml) 87.9
33 C Caffeine (10 mg/ml)/ 66.1 49 Arginine (25 mg/ml) D Arginine
(25 mg/ml) 76.7 41
[0110] Arginine was added to 280 mg/mL solutions of BGG in
histidine buffer at pH 6. At levels above 50 mg/mL, adding more
arginine did not decrease viscosity further, as shown in Table
16.
TABLE-US-00016 TABLE 16 Arginine added (mg/mL) Viscosity (cP)
Viscosity reduction (%) 0 79.0 0% 53 40.9 48% 79 46.1 42% 105 47.8
40% 132 49.0 38% 158 48.0 39% 174 50.3 36% 211 51.4 35%
[0111] Caffeine was added to 280 mg/mL solutions of BGG in
histidine buffer at pH 6. At levels above 10 mg/ml, adding more
caffeine did not decrease viscosity further, as shown in Table
17.
TABLE-US-00017 TABLE 17 Caffeine added (mg/mL) Viscosity (cP)
Viscosity reduction (%) 0 79 0% 10 60 31% 15 62 23% 22 50 45%
EQUIVALENTS
[0112] While specific embodiments of the subject invention have
been disclosed herein, the above specification is illustrative and
not restrictive. While this invention has been particularly shown
and described with references to preferred embodiments thereof, it
will be understood by those skilled in the art that various changes
in form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims. Many
variations of the invention will become apparent to those of
skilled art upon review of this specification. Unless otherwise
indicated, all numbers expressing reaction conditions, quantities
of ingredients, and so forth, as used in this specification and the
claims are to be understood as being modified in all instances by
the term "about." Accordingly, unless indicated to the contrary,
the numerical parameters set forth herein are approximations that
can vary depending upon the desired properties sought to be
obtained by the present invention.
* * * * *